Autoregulation.

Autoregulation of CBF refers to the maintenance of a constant CBF over a broad range of perfusion pressures. This constancy of CBF results from arteriolar vasoconstriction with increased perfusion pressure and vasodilation with decreased perfusion pressure. The mechanisms underlying autoregulation are not entirely understood. Currently the balance of data suggests that autoregulation is mediated primarily by an interplay between endothelial-derived constricting and relaxing factors (see the later discussion of perinatal CBF). Autoregulation in the adult human is operative over a range of mean blood pressure between approximately 60 and 150 mm Hg, and the response time is approximately 3 to 15 seconds.

Coupling of Cerebral Function, Metabolism, and Blood Flow.

Tight coupling of cerebral function, metabolism, and blood flow is well established and can be demonstrated by a variety of correlative physiological, biochemical, and even clinical studies. This coupling appears to be mediated by regulation of CBF by one or more local chemical factors that are vasoactive. Vasoactive factors of importance in the brain include H ⁺ , K ⁺ , adenosine, prostaglandins, osmolarity, and Ca ²⁺ ( Table 13.2 ). Increase in the perivascular H ⁺ concentration (i.e., decrease in local pH) is associated with arteriolar vasodilation. Greater neuronal metabolic activity can decrease local pH and therefore increase substrate supply. The effect of perivascular H ⁺ concentration mediates the vasodilating action of arteriolar carbon dioxide and is important under a variety of other physiological and pathological conditions (see later discussion). This vascular response is well established in the perinatal brain (see later section). K ⁺ has a vasoactive effect. Vasodilation increases linearly with extracellular K ⁺ levels to 10 mmol/kg (levels >20 mmol/kg induce vasoconstriction). The vasodilation is mediated by a Ca ²⁺ -activated K ⁺ channel on vascular smooth muscle. Because K ⁺ is released from nerve cells with electrical activity or a variety of insults, including oxygen deprivation (see earlier discussion), this ion may play a role in the regulation of CBF under certain pathological conditions.

Adenosine , administered on the perivascular side of pial arteries, results in a concentration-dependent vasodilation. Changes in adenosine also accompany certain pathological states with changes in CBF, including oxygen deprivation. Prostaglandins , particularly prostaglandins E and F ₂ , lead to cerebral vasodilation. The concentrations of these compounds increase in response to cerebral ischemia, and agents that inhibit prostaglandin biosynthesis (e.g., indomethacin) have cerebral vasoconstrictive effects. Prostaglandins appear to be of cerebral hemodynamic importance in immature animals (see later discussion). Increases in perivascular osmolarity have a vasodilating effect, and decreases have a vasoconstricting effect. These data may bear on such effects on CBF as the vasodilation associated with infusion of hypertonic solutions. Ca ²⁺ may also play a role in the control of CBF (e.g., high perivascular concentrations of Ca ²⁺ lead to vasoconstriction, and low concentrations lead to dilation of cerebral vessels). The ability of Ca ²⁺ channel blockers to lead to increases in CBF relates to the prevention of the vasoconstricting effects of Ca ²⁺ . Extracellular Ca ²⁺ concentrations decline with hypoxia (see earlier discussion) and status epilepticus. Other chemical factors (e.g., renin-angiotensin, vasopressin, endogenous opioids, other neuropeptides, adrenergic compounds, acetylcholine, and endothelium-derived relaxing and constricting factors) may play important roles in the regulation of CBF, but more data are needed on these issues. Limited data are available on these agents in immature animals. One report shows that morphine infusions in neonatal piglets result in an upregulation of the vasoconstrictor, endothelin-1, and its receptors.

Ontogenetic Effects.

Total and regional CBF changes significantly with maturation. In general, CBF overall increases with postnatal age, and this increase correlates well with similar increases in cerebral metabolic rates and energy demands, and with neuroanatomical development. Changes in regional CBF with maturation also reflect coupling with metabolic and anatomical development . The most dramatic short-term ontogenetic change in CBF occurs around the time of birth. In the lamb, CBF decreases by approximately threefold in the first 24 hours after birth. This decrease correlates well with the postnatal increase in oxygen content ( Fig. 13.7 ), consistent with the importance of oxygen delivery in the regulation of CBF.

Correlation of the perinatal decrease in cerebral blood flow with the increase in arterial oxygen content in the lamb.

(From Richardson BS, Carmichael L, Homan J, Tanswell K, Webster AC. Regional blood flow change in the lamb during the perinatal period. Am J Obstet Gynecol . 1989;160:919–925.)

Regional Effects.

Impressive regional differences in CBF are apparent in the perinatal animal, and these differences relate in considerable part to regional differences in metabolic activity. Using the microsphere technique to study regional CBF in term fetal sheep, Ashwal et al. initially noted (1) higher flows in brain stem than in cerebrum and (2) higher values in cortical gray matter than in subcortical white matter. Cavazutti and Duffy amplified these findings in a study of blood flow to 32 brain regions in the newborn dog, confirming the highest CBF in cerebral gray matter, nuclear structures of the brain stem, and diencephalon and lowest in cerebral white matter. Blood flows to the cerebral cortex were approximately 5- to 10-fold of those to subcortical white matter. These regional differences have been confirmed in studies of the perinatal rabbit, lamb, piglet, and puppy. Parallel studies of regional CBF and cerebral glucose metabolism ([2- ¹⁴ C]deoxyglucose method) demonstrated a close correlation with CBF, thus indicating that the coupling of blood flow and metabolism is present in the neonatal and adult animal. Finally, studies of blood flow to various regions of primate cerebrum indicate that the parasagittal regions , especially in the posterior aspects of the cerebral hemispheres, have significantly lower flow than other cerebral regions. This finding may have major implications for the distribution of brain injury with perinatal ischemic insults (see the section on parasagittal cerebral injury in Chapter 20 ).

Cerebral Autoregulation.

Cerebral autoregulation appears to be operative over a broad range of arterial blood pressure in the preterm and term fetal lamb, the neonatal lamb, and the neonatal dog. The principal stimulus for the autoregulatory change in vascular diameter appears to be induced largely by the deformation of endothelial cells and generation of endothelial-derived signals that act on the vascular smooth muscle. With a decrease in transmural pressure, NO- and Ca ²⁺ -activated K ⁺ channels are important in the vasodilation response, and with an increase in transmural pressure, endothelin-1 is critical in mediation of the vasoconstriction response. The autoregulatory range of blood pressures varies slightly among species and experimental conditions. The curve for the preterm lamb at approximately 80% gestation is shown in Fig. 13.8 . The curve for the preterm lamb differs from that for the term or neonatal lamb in two respects. First, the autoregulatory range in the preterm lamb is narrower , especially at the upper limit of the curve. Second, and perhaps more strikingly, the normal arterial blood pressure in the preterm lamb is very near or at the lower autoregulatory limit . Indeed, in the preterm lamb at 80% of gestation, normal arterial blood pressure is only 5 to 10 mm Hg above the lower limit of the curve, in contrast to the situation in older animals. In a subsequent study that included preterm fetal lambs at approximately 65% gestation (i.e., the onset of the third trimester), the lower autoregulatory limit was essentially identical to the normal resting arterial blood pressure. More recent data indicate that the range of blood pressure over which autoregulation is operative decreases with lower gestational age. These data indicate that with decreasing gestational age, resting mean arterial blood pressure (MABP) values approach the lower limit of the autoregulatory plateau, and the range of blood pressure over which CBF remains constant narrows . Stated in another way, the observations suggest that the margin of safety, at least in the preterm fetus, and to a lesser extent in the term fetus, is small at the lower end of the autoregulatory curve and points to vulnerability to ischemic brain injury with modest hypotension , particularly in the preterm animal. Vulnerability to hypertension also may result because little change occurs in the upper limit of the autoregulatory range during a brief developmental period (third trimester in the lamb and the human) when normal arterial blood pressure increases markedly. Thus normal arterial blood pressure shifts precariously close to the upper autoregulatory limit and renders capillary beds (e.g., germinal matrix) vulnerable to hemorrhage with modest hypertension.

Autoregulation of cerebral blood flow in the preterm lamb. See text for details.

(From Papile LA, Rudolph AM, Heymann MA. Autoregulation of cerebral blood flow in the preterm fetal lamb. Pediatr Res . 1985;19:159–161.)

Autoregulation in the term fetal lamb and in the newborn lamb has been shown to be sensitive to hypoxia . Changes in PaO ₂ from 20 to 16 mm Hg in the fetal animal and from approximately 70 to 30 mm Hg in the newborn animal abolished autoregulation. These decreases in PaO ₂ resulted in decreases in arterial oxygen saturation of less than 50%, which can be considered a hypoxic threshold for impairment of cerebrovascular autoregulation. The impairment of autoregulation required only a 20-minute exposure to hypoxia, and autoregulation did not recover until 7 hours after restoration of normoxia . Studies in adult animals showed that autoregulation is abolished in the presence of hypercarbia , and a similar phenomenon was observed in the perinatal animal and in the human preterm newborn. In a single study of the newborn lamb, systemic acidosis also was shown to cause a loss in cerebrovascular autoregulation .

Regional variation in the decrease in CBF provoked by hypotension to blood pressure values below the lower limit of the autoregulatory plateau has been described in the neonatal piglet, puppy, and lamb. In the neonatal piglet, the percentage of reduction in blood flow was least to the brain stem and greatest to the cerebrum. In a more detailed regional study in the newborn puppy, the flow to cerebral white matter was most vulnerable to hypotension . Similarly, in the preterm lamb, the lower autoregulatory limit with hypotension was lower in the brain stem than in the cerebrum. Perhaps even more important, in the preterm lamb at the start of the third trimester, blood flow to cerebral white matter not only was particularly vulnerable to hypotension but also did not recover under conditions of reperfusion that restored blood flow to all other brain regions ( Figs. 13.9 and 13.10 ). The latter observations may have implications for the topography of the injury in the immature human brain with hypoxic-ischemic insults (see the following discussion and Chapter 14 , Chapter 15 , Chapter 16 ).

Mean arterial blood pressure and regional cerebral blood flow (CBF; percentage of control values, mean ± SE) after hemorrhagic hypotension and reinfusion in the preterm lamb .

The approximately 25% reduction in blood pressure resulted in significant lowering of CBF in all regions. However, only in cerebral white matter CBF failed to return to baseline levels on reinfusion.

(From Szymonowicz W, Walker AM, Yu VY, Stewart ML, Cannata J, Cussen L. Regional cerebral blood flow after hemorrhagic hypotension in the preterm, near-term, and newborn lamb. Pediatr Res . 1990;28:361–366.)

Mean arterial blood pressure and regional cerebral blood flow (CBF; percentage of control values, mean ± SE) after hemorrhagic hypotension and reinfusion in the near-term lamb.

In contrast to the effect on CBF of a similar decrease in blood pressure in the preterm lamb (see Fig. 13.9 ), during hypotension a significant decline occurred only in cerebral cortex, and on reinfusion all values returned to baseline levels.

(From Szymonowicz W, Walker AM, Yu VY, Stewart ML, Cannata J, Cussen L. Regional cerebral blood flow after hemorrhagic hypotension in the preterm, near-term, and newborn lamb. Pediatr Res . 1990;28:361–366.)

Changes in arterial PCO ₂ (i.e., PaCO ₂ ) have marked effects on CBF in perinatal as in adult animals. In a study of blood flow to 32 brain regions of the newborn dog, a positive linear correlation was obtained in each structure examined. However, the response to carbon dioxide varied widely among brain regions, ranging from an increase of only 0.15 mL/100 g per minute per mm Hg in PCO ₂ in subcortical white matter to an increase of 4.8 in the vestibular and superior olivary nuclei. The limited vasodilatory response in cerebral white matter may have implications for the vulnerability of this region to hypoxic-ischemic injury. In general, the higher the blood flow to a particular structure, the greater the vasodilatory response to increasing PCO ₂ ( Fig. 13.11 ).

Sensitivity of local cerebral blood flow to arterial partial pressure of carbon dioxide (PaCO ₂ ).

Note that regions with the higher normocapnic blood flow exhibit the highest sensitivities to PaCO ₂ .

(From Cavazutti M, Duffy TE. Regulation of local cerebral blood flow in normal and hypoxic newborn dogs. Ann Neurol . 1982;11:247–257.)

The effects of profound hypocarbia on CBF are also of importance , as it occurs frequently in sick infants requiring substantial respiratory support. Studies in neonatal lambs indicated an abrupt decrease in CBF with hypocarbia induced by hyperventilation. The decrease was nonlinear, such that the vasoconstricting effect of hypocarbia declined at lower PaCO ₂ tensions. Moreover, the decline in CBF became less prominent with time, such that CBF was no longer statistically different in severely hypocarbic animals (PaCO ₂ = 15 mm Hg) compared with control animals (PaCO ₂ = 36 mm Hg) after 6 hours ( Table 13.3 ). Perhaps most important, the declines in CBF did not cause any change in cerebral metabolic rate for oxygen because cerebral oxygen extraction fraction increased. The attenuation of the decline in CBF with increasing duration of hypocarbia probably relates to an increase in perivascular H ⁺ concentration, primarily secondary to an increase in lactate levels. This formulation is supported by the marked increase in CBF above baseline levels on restoration of normocarbia.

The effects of modest hypocarbia (e.g., PaCO ₂ = 26 mm Hg) interact adversely with hypoxic-ischemic insults in the 7-day-old animal. Thus animals exposed to hypoxia-ischemia sustained a greater decline in CBF and a greater degree of brain injury when they were simultaneously rendered modestly hypocarbic, compared with animals who were normocarbic (PaCO ₂ = 39 mm Hg) or rendered mildly hypercarbic (PaCO ₂ = 54 mm Hg). The latter two groups had better preserved CBF, less severe cerebral metabolic deficits, and less severe brain injury. The modestly hypercarbic animals had the most favorable hemodynamic, biochemical, and neuropathological outcomes. However, more marked hypercarbia accentuated brain injury, because of marked decrease in CBF. The latter effect is presumably related to impaired autoregulation and cardiovascular depression.

Alterations in arterial PO ₂ (PaO ₂ ) also cause distinct changes in CBF. ^a

a References .

The role of acidemia in the regulation of CBF in the perinatal animal requires further study. Whether produced by hypoxemia, lactate infusion, or respiratory means, acidemia caused a sharp increase in CBF in perinatal goats. However, effects were most impressive when acidemia was induced by elevation in PaCO ₂ , a potent effector of CBF (as described earlier). Moreover, a subsequent study of the fetal lamb did not report an alteration in regional CBF over an arterial pH range from 6.9 to 7.5 produced by infusions of lactate or bicarbonate. Studies of newborn dogs and piglets showed inconsistent effects of lactate infusions or other changes in arterial pH on CBF.

A role for adenosine in regulation of CBF in the immature brain is suggested by observations, primarily in the neonatal piglet. Studies correlated CBF with parallel measurements of interstitial concentrations of adenosine and have used specific agonists and antagonists of the A ₂ receptor (the adenosine receptor on vascular smooth muscle; the A ₁ receptor, the adenosine receptor on neurons, is involved in decreasing glutamate release and Ca ²⁺ influx). The data suggest that adenosine has a vasodilatory effect and that it is involved in the cerebrovascular response to decreases in blood pressure and thereby cerebrovascular autoregulation. Recall that brain adenosine concentrations increase with hypoxia and seizures; both conditions require increases in substrate influx to brain. Finally, in addition to its vasodilatory effect, adenosine may influence CBF by inhibitions of platelet aggregation and activation of neutrophils (implicated in endothelial dysfunction), events shown to be important in the postischemic impairment of CBF in adult models.

Prostaglandins are important regulators of CBF in the perinatal period. ^a

a References .

Redistribution of Fetal Circulation.

Promptly after the onset of asphyxia in the term fetal primate or lamb, cardiac output is redistributed such that a significantly larger proportion enters the brain, the coronary circulation, and the adrenals, at the expense of blood flow to other regions. ^a

a References .

Increase in Cerebral Blood Flow.

The major purpose of the circulatory changes as outlined is to maintain CBF in the presence of impending tissue oxygen debt. In experiments with fetal and neonatal lambs, puppies, and primates, CBF in perinatal asphyxia increased generally by 50% to 500%. ^b

b References .

The mechanisms underlying the initial increase in CBF relate in part to cerebral vasodilation, secondary to hypoxemia or hypercapnia, or both, presumably with increased perivascular H ⁺ concentration. Roles for elevated extracellular fluid concentrations of K ⁺ , adenosine, and prostaglandins, all of which increase markedly in brain with hypoxemia and ischemia, are likely. The particular importance of a rise or at least maintenance of blood pressure in the increase of CBF with asphyxia was indicated by several studies. In term fetal sheep subjected to asphyxia by cord compression, the initial increase in MABP persisted for 60 minutes before decreasing to normal values. Carefully controlled experiments with the same animal suggested that fetal blood pressure may be even more critical than local chemical factors, which lead to cerebral vasodilation, in the enhancement of CBF.

Although blood flow to various regions of the brain increases generally in concert with the increase in total CBF, distinct regional differences in this increase are apparent. In general, the increase in blood flow is most marked in brain stem structures and is least apparent in cerebral white matter. This general pattern was documented in the fetal lamb, neonatal lamb, and neonatal puppy. ^c

c References .

Loss of Vascular Autoregulation.

A serious impairment of cerebral vascular autoregulation develops with perinatal asphyxia with the presence of a pressure-passive CBF. Using the radioactive microsphere technique and producing asphyxia (pH. 6.8 to 7.0) in term fetal sheep by partial occlusion of umbilical vessels, Lou et al. demonstrated a striking pressure-passive CBF. Marked hyperemia, with CBF values up to six times the normal value, occurred when MABP was raised to 60 to 70 mm Hg, whereas CBF declined to close to zero in large cortical areas when MABP was lowered to 30 mm Hg. Vascular autoregulation in these term fetal animals appeared to be very sensitive to asphyxia. The likely mechanism relates most probably to the hypoxemia and hypercapnia that are the hallmarks of perinatal asphyxia. The sensitivity of the autoregulatory system in the fetal and neonatal brain to these alterations in blood gas levels was described earlier (see the section on autoregulation). The implications of these data for ischemic injury to perinatal brain are obvious. In one study of near-term fetal sheep, autoregulation of CBF was lost within 4 minutes of cord occlusion, and overt cerebral injury occurred by 10 minutes secondary to decreased CBF in the presence of hypotension.

Hypotension and Diminished Cerebral Blood Flow.

Although the initial response to asphyxia is hypertension, this response is followed by hypotension. The rapidity and severity of this occurrence depend on the duration and severity of the asphyxial insult. In large part, this effect is related to a diminution in cardiac output, probably secondary to an effect on the myocardium. The consequence for the brain may be devastating, because the impairment of vascular autoregulation leaves CBF at the mercy of perfusion pressure. Deficits in CBF may be marked, with relatively modest changes in MABP. Impressive deficits in CBF (20% to 80%) have been demonstrated, particularly in the parasagittal regions of the cerebral hemispheres and especially posteriorly, in the term fetal monkey subjected to severe and prolonged asphyxia. A similar parasagittal distribution of cerebral cortical injury was demonstrated in near-term fetal sheep subjected to cerebral ischemia. The detailed regional study of newborn dogs demonstrated that cerebral white matter also is particularly likely to exhibit diminished blood flow with hypotension. In the preterm sheep (0.65 gestation), cerebral white matter was particularly affected with ischemia, with white matter injury then determined by the topographic distribution within the ischemic regions of vulnerable differentiating oligodendrocytes. These observations correlate well with the neuropathological and clinical observations made by asphyxiated human infants (see Chapters 16 and 20 ).

A summary of the major relationships between perinatal asphyxia and CBF during asphyxia is shown graphically in Fig. 13.12 . The initial effects leading to increased CBF are considered best as compensatory, adaptive responses (which could become maladaptive by leading to hemorrhage in vulnerable capillary beds). The later effects represent a decompensation of these responses and a cascade that leads to diminished CBF and brain injury.

Major relationships between perinatal asphyxia and cerebral blood flow (CBF).

The major consequences of the changes in cerebral blood flow (i.e., hemorrhage and ischemic brain injury) are shown. BP , Blood pressure.

Postasphyxial-Postischemic Effects.

The period after termination of the asphyxial-ischemic insult is critical because, during this interval, progression to brain injury occurs (see earlier discussion), and in the clinical setting, this time represents a window of opportunity for therapeutic intervention. The principal experimental models used have been near-term fetal sheep and neonatal piglets and rat pups, and the insults have primarily consisted of hypoxia-ischemia and, less commonly, asphyxia. ^a

a References .

Cerebral hemodynamic changes after transient cerebral ischemia in the late-gestation fetal lamb.

Changes in total cerebral hemoglobin (tHb), oxygenated hemoglobin (HbO ₂ ), and cytochrome oxidase (CytO ₂ ) were measured by near-infrared spectroscopy. In each graph, different symbols represent data from separate fetuses. Note the two phases of cerebral vasodilation and increased cerebral blood flow, as assessed by the hemoglobin signals; the early increase occurs in the first 2 to 3 hours after ischemia, and the delayed increase occurs from 12 to 48 hours. The delayed increase in flow is accompanied by a decline in CytO ₂ , consistent with impaired mitochondrial oxygenation.

(From Marks KA, Mallard EC, Roberts I, et al. Delayed vasodilation and altered oxygenation after cerebral ischemia in fetal sheep. Pediatr Res . 1996;39:48–54.)

Postasphyxial impairment of cerebrovascular autoregulation.

Cerebral blood flow versus mean arterial blood pressure following asphyxia. Symbols ( n = 7) represent responses to changes in blood pressure in individual asphyxiated lambs ( n = 7). The regression line is derived from pooled data of all lambs. The dashed line represents data from nonasphyxiated (control) lambs.

(From Rosenberg AA. Regulation of cerebral blood flow after asphyxia in neonatal lambs. Stroke. 1988;19:239–244.)

Importantly, a second (i.e., “delayed”) increase in CBF develops, with onset generally between 12 and 24 hours and with a duration of many hours or a day or more (see Fig. 13.13 ). This increase is associated with evidence of impaired mitochondrial oxygenation (as assessed by brain levels of oxidized cytochrome c), with the cellular energy failure, and with neuropathological evidence for neuronal and white matter injury. The delayed hyperemia, its association with energy failure, and its correlation with severity of brain injury have also been observed in asphyxiated human infants ( Chapter 18 , Chapter 19 , Chapter 20 ). The mechanisms underlying the vasodilation and hyperemia are not established but may relate in part to NO. Because NO synthesis in endothelial cells (eNOS) is activated after hypoxic-ischemic insults, an attempt to reduce the delayed cerebral hyperemia with an NOS inhibitor was attempted in the fetal sheep model of ischemia. The inhibitor did attenuate the delayed hyperemia, but surprisingly, inhibition of NO synthesis increased histological injury. This combination of findings suggests that NO synthesis has a protective effect either because of the vasodilatory effect or because of a biochemical effect.

Changes Immediately After Delivery.

A sharp decrease in “apparent” CBF (“apparent” because the plethysmographic method is only semiquantitative) occurs in the term infant in the first hours after delivery ( Fig. 13.16 ). The decrease in the first 3 hours is nearly twofold, and over the ensuing hours, CBF is relatively stable. The reason for the relatively higher value shortly after delivery is not known, although a relationship with higher PaCO ₂ levels immediately after birth has been suggested. Alternatively, it is possible that a reflex activity, mediated by vagal afferents, is operative because the relatively higher CBF near the time of birth requires intact vagus nerves in the sheep. Both factors may be relevant in the human newborn, when PaCO ₂ levels may be elevated and vagal activity from lung expansion may be considerable. A third factor may involve arterial oxygen content, because, as noted earlier, a similar sharp decline in CBF after birth has been observed in the lamb and correlates well with the increase in arterial oxygen content in the newborn versus fetal state. The relatively enhanced CBF in the first minutes to hours after delivery may provide a margin of safety for cerebral metabolic needs in the period of adaptation to birth.

Apparent cerebral blood flow in term infants following delivery, as estimated by the jugular venous occlusion plethysmographic technique.

Note the decline in the first several hours, followed by stable flow.

(From Cooke RW, Rolfe P, Howat P. Apparent cerebral blood-flow in newborns with respiratory disease. Dev Med Child Neurol . 1979;21:154.)

Changes Beyond the Immediate Postpartum Period.

Data obtained by PET suggest that CBF is approximately 20% of the adult value in the premature infant of approximately 28 weeks of gestation and approximately 40% of the adult value in the term newborn. Serial studies of CBF in normal preterm infants show an approximately twofold increase in flow over the first 3 days of life, perhaps related to an increase in cardiac output. One study of preterm infants by spatially resolved near-infrared spectroscopy showed a sharp increase in cerebral oxygenation during this period, most consistent with an increase in CBF. Doppler studies of CBF velocity also are consistent with this postnatal increase in CBF (see Chapter 10 ).

Normal Values.

Values for CBF reported in the human premature newborn, studied by xenon clearance and shown in Table 13.7 , are generally between 10 and 20 mL/100 g per minute. A similar range is apparent in studies by PET and near-infrared spectroscopy. The correlations with the findings in developing animals described earlier are obvious. Regional values for CBF are notable for higher flows in cerebral gray matter structures than in cerebral white matter.

Regulation in the Human Newborn.

The major established regulatory mechanisms for CBF in the human newborn include autoregulation, PaCO ₂ , oxygen delivery, blood glucose, and neuronal activity (e.g., seizure). Certain pharmacological agents also have been shown to exert regulatory effects. These various regulatory factors and their effects on CBF are summarized in Table 13.8 and are reviewed briefly next.

Autoregulation.

Autoregulation appears to be operative in both the normal human preterm and full-term infants. ^a

a References .

b References .

An approximation of the relationship of cerebral blood flow as a function of mean arterial blood pressure (MABP) with maturation. See text for details.

The problem of a persistent pressure-passive cerebral circulation is particularly important in sick premature infants. Thus initial studies using the invasive technique of radioactive xenon clearance initially showed that certain premature infants, mechanically ventilated and usually clinically unstable, appeared to exhibit pressure-passive cerebral circulation. This fundamental initial observation has been confirmed by less invasive methods multiple times. Thus, in such sick premature infants with pressure-passive cerebral circulation, it would be expected that when blood pressure falls, as occurs commonly in such infants, so would CBF, with the consequence of cerebral ischemia. The presence of arterial end zones and border zones and vulnerable early differentiating oligodendroglia in developing cerebral white matter would render this region especially vulnerable (see Chapter 15 ). Moreover, the particular danger is compounded by the very low normal blood flow to cerebral white matter in the premature infant, a feature suggesting that a minimal margin of safety may exist. In one serial study of 32 mechanically ventilated premature infants from the first hours, near-infrared spectroscopy was used to demonstrate a pressure-passive cerebral circulation in 53%. The example shown in Fig. 13.18 is typical (i.e., an infant with MABPs that fluctuate gradually over minutes and are accompanied by parallel changes in the cerebral circulation). The nadirs of blood pressure often are not markedly low and thus may be readily overlooked . However, the cumulative effect of many repeated modest declines in CBF is likely to be considerable (see Chapter 15 ). Importantly, nearly all the cases of PVL (and severe germinal matrix hemorrhage/intraventricular hemorrhage) later identified in the series of 32 infants were in the pressure-passive group. These findings were critical because they suggested that (1) infants with a pressure-passive cerebral circulation could be identified by near-infrared spectroscopy before the occurrence of white matter injury, (2) that the circulatory abnormality is related strongly to the occurrence of white matter injury, and (3) if the pressure-passive state could be corrected, perhaps the white matter injury could be prevented. However, this study involved a relatively small number of infants.

Pressure-passive relationship of cerebral blood flow, estimated by near-infrared spectroscopy, and mean arterial blood pressure, in ventilated premature infants.

Note the parallel changes in arterial blood pressure and cerebral perfusion (A–C). Note also that the changes in blood pressure are not marked. In (C) all data points are plotted; the linear relationship between blood pressure and cerebral perfusion is apparent. CBF , Cerebral blood flow; MABP , mean arterial blood pressure.

(From Tsuji M, Saul JP, duPlessis A, et al. Cerebral intravascular oxygenation correlates with mean arterial pressure in critically ill premature infants. Pediatrics. 2000;106:625–632.)

A subsequent study of 90 premature infants used a frequency-based assessment of autoregulation and quantitated the degree and duration of altered autoregulation over the first 5 days of life. Pressure-passive epochs were documented in 95% of the infants . The overall mean proportion of the pressure-passive time was 20%, although some infants had a pressure-passive state more than 50% of the time. The likelihood of a pressure-passive state increased with decreasing gestational age and periods of hypotension.

More recently, using near infrared spectroscopy (NIRS) methodology, high coherence between tissue oxygenation index (TOI) and MABP was also observed with low blood pressure in the sickest infants, suggestive of a narrow window for autoregulation in such sick immature infants. Additional technical analysis with time domain analysis described the cerebral oximetry index (COx) as the moving and linear correlation coefficient between slow waves of MABP and regional cerebral oxygen saturation (rSO ₂ ). Researchers observed that lower MABP during the first 3 days of life was associated with impaired autoregulation (COx >0.5) in preterm infants at ≤30 weeks of gestational age. There has been debate regarding whether the two methods are equivalent or one is superior to the other; however, in a series of 60 preterm infants, time-domain analysis appeared more robust compared with coherence function analysis.

A further index of cerebral vascular reactivity is a correlation coefficient of TOI and MABP (TOx) with HR (tissue oxygenation heart rate, TOHRx), believed to improve the reflection of perfusion with both blood pressure and heart rate. Using this measure in 31 preterm infants at median gestational age of 26 weeks, TOHRx was significantly correlated with gestational age ( R = −0.57, P = .007), birth weight ( R = −0.58, P = .006), and the Clinical Risk Index for Babies II ( R = 0.55, P = .0014)—a prediction score of mortality and morbidity in the preterm infant. Defining “optimal” MABP by the strength of cerebral autoregulation has been studied in adults with traumatic brain injury. By measuring spontaneous fluctuations in cerebral perfusion pressure, it is possible to calculate the range of perfusion pressure where autoregulation is strongest. In one study, patients were more likely to have a favorable outcome when cerebral perfusion pressure was close to “optimal.” Recently, continuous monitoring of cerebral vascular reactivity in preterm infants, with TOHRx, has been used to define values of MABP _OPT where cerebral vascular reactivity is strongest. Preterm infants who died had a higher mean absolute deviation from MABP _OPT than patients who survived. Furthermore, deviation of MABP above the optimal values was observed in infants who had worse germinal matrix-intraventricular hemorrhage grades. (There remain significant technical challenges in obtaining reliable measurements of MABP _OPT at the cot side. For example, a minimum of 2 to 4 hours of continuous artifact-free data is necessary to create a reliable U -shaped autoregulatory curve with spontaneous fluctuations in heart rate and TOI. Currently all descriptions of MABP _OPT have been undertaken on retrospective analysis of data.)

The lower limit of the autoregulatory curve in newborns obviously is of great importance, particularly for management purposes and especially in sick premature infants. The precise lower limit remains unknown, and it is likely that this value varies not only with gestational age but also with postnatal age and with factors that alter the concentration of vasoactive molecules in brain (e.g., blood gases, cytokines, seizures, hypoxia-ischemia). In one report, blood pressure values in the first day of life in infants 24 to 30 weeks of gestation of less than 31 mm Hg were associated with impaired electroencephalogram (EEG) continuity on the EEG, a finding raising the possibility of decreased CBF at such values. Similarly, in a study of extremely low-birth-weight infants (mean birth weight 772 g, mean gestational age 26 weeks) by near-infrared spectroscopy, a pressure-passive cerebral circulation resulted when MABPs were lower than 30 mm Hg. However, to what extent, if any, arterial blood pressures lower than 30 mm Hg are clearly dangerous is unclear. Thus a careful study of cerebral electrical activity and cerebral fractional oxygen extraction, presumably a reflection of CBF, in 35 23- to 30-week premature infants showed no electrical abnormality at mean blood pressure levels higher than 23 mm Hg.

Carbon Dioxide.

PaCO ₂ is a potent regulator of CBF in the human newborn. ^a

a References .

The mechanism for the vasodilating effect of carbon dioxide relates to the increase in perivascular H ⁺ concentration , as observed in experimental models. The observation of a 50% decrease in CBF after sodium bicarbonate administration to term and premature infants with acidosis also supports the important role of perivascular H ⁺ concentration. The mechanism proposed for a decrease in the latter situation was enhanced movement of bicarbonate across the blood-brain barrier “because of vasodilation caused by the asphyxia” in these infants. The demonstration of a decrease in CBF after sodium bicarbonate administration in acidotic postasphyxial infants may have important implications for management. Sodium bicarbonate is no longer recommended for use in the resuscitation of the term or preterm newborn infant due to its association with an increased mortality and morbidity.

Oxygen.

Arterial oxygen concentration is an important effector of CBF in the human infant, as it is in the perinatal animal. ^a

a References .

Glucose.

A striking observation by Pryds et al. established an important role for glucose in regulation of CBF in the human newborn (see Table 13.8 ). As discussed in more detail in Chapter 25 , an increase in CBF became apparent as blood glucose concentration decreased to less than approximately 30 mg/dL (1.7 mmol/L). Increases in CBF of twofold to threefold then occurred in proportion to the decline in blood glucose. The mechanism for this vasodilatory effect of glucose is not clear, but stimulation of beta-receptors by the increased compensatory secretion of epinephrine is suggested by data in human adults. The clinical significance of this effect of glucose could be appreciable (see Chapter 25 ).

Neuronal Activity (Seizure).

The coupling of neuronal activity to CBF is apparent in at least two situations: sleep states and seizure. A decrease in CBF during sleep has been shown by xenon-133 clearance. The effect is not striking. A striking increase (≈50%) in CBF with the excessive neuronal activity of seizure has been documented in the human newborn by PET (see Chapter 12 ). This effect had been suggested by earlier studies of CBF velocity by Doppler.

Pharmacological Agents.

Indomethacin and aminophylline are the two pharmacological agents shown to have a clear effect on CBF in the human newborn. Indomethacin administration in doses used for closure of the ductus arteriosus led to a 20% to 40% decrease in CBF in the premature infant, as studied by xenon-133 clearance and near-infrared spectroscopy. Notably, however, no change in CBF, assessed by Doppler, was observed after continuous versus bolus infusion of indomethacin. The vasoconstrictive effect is mediated by the inhibition of synthesis of vasodilatory prostaglandins at the cyclooxygenase step, as shown in experimental models and studies of isolated neonatal human cerebral artery. An increase in cerebrovascular resistance was shown by Doppler studies of very low-birth-weight infants after indomethacin administration. Interestingly, by contrast with indomethacin, ibuprofen does not lead to a decline in CBF.

Administration of aminophylline , an antagonist of adenosine, led to only a small decrease (10% to 15%) in CBF in the human premature infant within 1 hour of intravenous administration. No alteration of visual evoked potentials accompanied this modest decrease in blood flow. The use of caffeine in the preterm infant has resulted in similar observations of a reduction in CBF. In a study of 40 preterm infants after a loading dose of caffeine using Doppler cerebral sonography, cardiac echocardiography, and cerebral spatially resolved near-infrared spectroscopy, mean anterior cerebral artery peak and time average mean blood flow velocity fell significantly (by 14% and 17.7%, respectively) at 1 hour post-caffeine loading dose. Cerebral TOI fell from predose levels by 9.5% at 1 hour, with partial recovery to 4.9% reduced at 4 hours post dose. There were no significant changes in left or right ventricular output, transcutaneous oxygen saturation, transcutaneous PCO ₂ , or total vascular resistance. Dopamine administered to treat hypotension was reported in two studies to increase CBF as well as arterial blood pressure. However, a more recent study of preterm and term piglets showed no increase of CBF with dopamine or dobutamine.

Impaired Autoregulation.

A xenon-133 study of CBF in a group of 19 term and preterm infants first suggested that autoregulation in the human newborn is very sensitive to perinatal asphyxia. Thus 19 infants were examined with the xenon clearance technique “a few hours after birth.” Eleven of these infants weighed less than 2000 g. Although most of the infants were considered “distressed,” Apgar scores at 5 minutes were less than 7 in only 4 of the 19 infants. At the time of study, pH was less than 7.20 in only 4 infants. For the total group of 19 infants, a linear relationship existed between CBF and systolic blood pressure ( Fig. 13.19 ). This pressure-passive relationship suggests inoperative vascular autoregulation and was seen to a similar degree in the infants less than or more than 2000 g body weight. This apparent impairment of vascular autoregulation is directly reminiscent of the data obtained with fetal and neonatal animals after asphyxia (see the earlier section).

Linear relationship between cerebral blood flow and systolic blood pressure in 10 newborns with Apgar scores of less than 7 at 1 minute.

Cerebral blood flow (CBF) was measured by the xenon clearance technique. A ₁ and A ₂ represent measurements of CBF in one patient before and after a spontaneous decrease in blood pressure. B ₁ and B ₂ represent measurements in another patient before and after a spontaneous increase in blood pressure.

(From Lou HC, Lassen NA, Friis-Hansen B. Low cerebral blood flow in hypotensive perinatal distress. J Pediatr . 1979;94:118.)

Impaired Vascular Reactivity and Cerebral Hyperemia.

Subsequent work clarified the relationship between perinatal asphyxia and the impairment of vascular reactivity—particularly autoregulation. In a systematic study of 19 term infants (mean birth weight, 3200 g) with perinatal asphyxia defined by a 5-minute Apgar score of 5 or lower and umbilical cord pH lower than 7.0, or both, a striking relationship among the severity of brain injury, the absolute value of CBF, and the reactivity to changes in blood pressure and PaCO ₂ was defined (see Table 13.10 ). Thus infants with the poorest neurological outcome (isoelectric amplitude-integrated EEG, death) had the highest values for CBF and no autoregulation or carbon dioxide reactivity (see Table 13.9 ). Infants with a burst-suppression EEG and moderate to severe brain injury had slightly elevated values for CBF and impaired autoregulation but retained reactivity to PaCO ₂ (see Table 13.10 ). Infants without evidence of brain injury had normal values for CBF, intact autoregulation, and reactivity to PaCO ₂ . A later study of 16 term infants with hypoxic-ischemic encephalopathy used PET to determine CBF primarily at 1 to 4 days of life and found higher flows in those infants with abnormal neurological outcome (35.6 mL/100 g per minute) than in those with normal neurological outcome (18.3 mL/100 g per minute).

The pronounced, sustained cerebral hyperemia observed in the human infant has been shown by less invasive techniques, such as Doppler ultrasound and near-infrared spectroscopy. Thus determinations of CBF velocity in term infants with hypoxic-ischemic encephalopathy from approximately 6 to 130 hours after the insult showed an increase in mean flow velocity with decreased resistance indices (i.e., vasodilation). Similarly, studies of asphyxiated human infants on the first day of life by near-infrared spectroscopy are consistent with a loss of vascular reactivity and an increase in cerebral blood volume and CBF, with temporal characteristics similar to those observed in fetal sheep, described earlier. The mechanism for this hyperemia is unclear. An increase in neuronal excitability, although documented after hypoxic-ischemic insults, seems unlikely in view of the relation of highest flows to isoelectric EEG. It appears more likely that an accumulation of vasodilatory compounds or vascular injury, or both, occurs and that this accumulation or injury in many ways may be similar in the human newborn and the perinatal animal. Delineation of the mechanisms underlying this vasoparalytic state and cerebral hyperemia after perinatal asphyxia in the human infant will be of major importance and may be able to be further explored with magnetic resonance techniques such as ASL.

The aforementioned data thus define in the postasphyxial human newborn a state of vasoparalysis and cerebral hyperemia that is correlated with the degree of brain injury and presumably the severity of the asphyxial insult. The altered vascular reactivity, with autoregulation impaired more readily than carbon dioxide reactivity, is similar to observations made in the postasphyxial state in perinatal animal models (see earlier discussion). Presumably, a state of maximal vasodilation exists, related perhaps to the effects of elevated perivascular H ⁺ concentration, prostaglandins, adenosine, free radicals, or NO, or all these factors. Whether this hyperemic state is caused by the same factors that lead to the brain injury, is an adaptive mechanism to preserve brain tissue, or in some way causes additional brain injury remains to be clarified. It does appear likely that the loss of vascular reactivity renders the infant vulnerable to systemic hypotension and resulting cerebral ischemia.

Maturational Vulnerability.

Studies of the effect of hypoxemia on brain energy metabolism in the immature rat brain have delineated a particular window of vulnerability. Thus the most marked declines in PCr and nucleoside triphosphates, defined by MR spectroscopy, occurred in the second postnatal week. This period of heightened vulnerability corresponds with the period of maximal susceptibility to excitotoxic neuronal injury and to epileptogenic effects of hypoxemia, as well as with the period of maximal expression of specific excitatory amino acid receptors, incomplete maturation of inhibitory transmission, relatively low levels of Ca ²⁺ binding proteins, and incomplete maturation of Na ⁺ /K ⁺ -ATPase levels (see later discussion). Taken together, these data suggest that the vulnerability of the immature rat in the second versus the first week of life relates to the increased propensity to develop with hypoxia, a hyperexcitable, hypermetabolic state in neurons, which leads to more marked declines in high-energy phosphates because of increased utilization. These considerations could help explain the greater likelihood of neuronal injury with hypoxia in the term brain than in the premature brain of the human.

Regional Vulnerability.

Studies in the newborn dog defined the regional changes in glucose and high-energy metabolism. Thus animals subjected to acute hypoxemia (oxygen pressure [PO ₂ ] ≈ 12 mm Hg) and studied by the autoradiographic 2-[ ¹⁴ C]deoxyglucose technique exhibited increased glucose utilization in most gray matter structures and every white matter structure. Moreover, the degree of hypoxemia was sufficient to cause the accumulation of lactate in the brain in both gray and white matter, but only in white matter did a decline in energy state occur ( Table 13.12 ). Thus it appears that anaerobic glycolysis with its accelerated glucose utilization was capable of preserving the energy state in gray matter but not in white matter . Moreover, the finding that glucose levels declined more drastically in white matter than in gray matter (see Table 13.12 ) suggests that glucose influx could not meet the increased demands for glucose in white matter. That the rate of glucose metabolism, in fact, was limited by glucose influx from blood is supported by the demonstration that local CBF increased insignificantly in relation to white matter but dramatically in relation to gray matter. The apparently limited vasodilatory capacity in white matter is discussed in the section on CBF, but this imbalance between glucose needs and glucose delivery may contribute to the propensity of neonatal cerebral white matter to hypoxic injury .

Importance of Endogenous Brain Glucose Reserves.

The biochemical mechanisms for the relation between carbohydrate status and resistance to hypoxic-ischemic insult relate to glycolytic capacity. Thus, with hypoxic-ischemic states, the replenishment of brain high-energy phosphate levels depends on anaerobic glycolysis. Because of the 19-fold reduction in ATP production per molecule of glucose when the brain is forced to oxidize glucose anaerobically, the glycolytic rate must be enhanced greatly. The adaptive mechanisms that come into play for this purpose are summarized in the previous sections. The greatly enhanced glycolytic rate leads to a decline of brain glucose levels. ^a

a References .

b References .

Greater importance of brain glucose reserves than glycogen in effects of hypoglycemia on vulnerability to anoxia (nitrogen breathing).

Brain glucose and glycogen levels in newborn rats were determined as a function of duration of anoxia. Hypoglycemia was produced by insulin injection at the onset of the experiment. At 60 minutes, survival was fivefold lower in hypoglycemic versus control animals (data not shown). At this 60-minute point, glucose was reduced much more severely than was glycogen. The arrow indicates subcutaneous administration of 10% glucose (1.8 g/kg). This resulted in a marked improvement in survival (data not shown) and a normalization of brain glucose but not of glycogen.

(From Vannucci RC, Vannucci SJ. Cerebral carbohydrate metabolism during hypoglycemia and anoxia in newborn rats. Ann Neurol . 1978;4:73–79.)

Summary.

Taken together, these data on immature animals (principally rodents) indicate that carbohydrate status plays an important role in determining the biochemical and clinical responses to hypoxemic and ischemic insults. Hypoglycemia is deleterious, and pretreatment with glucose is beneficial. The mechanism of the effect appears to relate to changes in endogenous, readily mobilized brain glucose reserves, which lead to the enhanced glycolytic rate required to slow the decline of, or even maintain the levels of, high-energy phosphate in the brain.

Importance of Severe Lactic Acidosis in Brain.

The biochemical mechanism for the deleterious effect of pretreatment with glucose in the previously mentioned juvenile monkeys may relate to the greater accumulation of lactic acid in the glucose-pretreated than in the food-deprived (control) monkeys ( Table 13.16 ). ATP levels declined approximately 10-fold in food-deprived animals subjected to circulatory arrest, and only a minimal difference in the magnitude of that decline was observed in animals pretreated with glucose. However, whereas lactate levels increased approximately fourfold in the food-deprived animals subjected to circulatory arrest, the levels increased more than 10-fold in those pretreated with glucose. The greater increases in brain lactate levels in the glucose-pretreated animals presumably reflected higher endogenous brain glucose reserves and, as a consequence, enhanced lactate production by anaerobic glycolysis. These experiments and related observations with animals rendered severely hypoxemic led Myers and Yamaguchi to suggest that the accumulation of brain lactate to concentrations of approximately 20 mmol/kg or greater leads to tissue destruction and brain edema. This approximate threshold level is supported by the observations that the accumulation of lactate to higher than this level occurs in thebrain of monkeys rendered ischemic in those regions that have been shown to be particularly vulnerable to neuronal injury.

Considerable support for the concept of a deleterious effect of abundant glucose and resulting lactic acidosis in brain in the pathogenesis of hypoxic-ischemic brain injury in the adult was provided by further studies in a variety of experimental models in mature animals . A threshold value of lactate of approximately 20 mmol/kg, above which major tissue injury occurs, can be suggested from the data. The apparent mechanism for the principal injury from these high levels of lactate is injury to endothelial cells, and perhaps also to perivascular astrocytes, with resulting disturbance of cerebral perfusion. Direct neuronal injury is likely, but widespread, secondary ischemic injury appears to develop primarily because of the vascular changes.

Summary.

Current experimental data allow several tentative conclusions to be made about the effects of glucose administration with perinatal hypoxic-ischemic insults ( Table 13.18 ). On balance, the findings favor maintenance of blood glucose concentrations in the normal range in infants who have sustained hypoxic-ischemic insults or who are at risk for sustaining such insults.

Mechanisms.

The mechanisms by which increased cytosolic Ca ²⁺ leads to cell death are multiple but are best discussed in the context of normal Ca ²⁺ homeostasis ( Fig. 13.34 ). The cellular mechanisms for maintenance of low cytosolic Ca ²⁺ concentrations (10 ⁻⁷ M) relative to high extracellular Ca ²⁺ concentrations (10 ⁻³ M) are located in the plasma membrane (voltage-dependent channels; three agonist-dependent, that is, glutamate-dependent, channels: the NMDA, AMPA [immature, i.e., lacking GluR2 subunit], and metabotropic receptor-activated channels [see later discussion]; an ATP-dependent uniport system, and an Na ⁺ -dependent antiport system), the endoplasmic reticulum (an ATP-dependent import system and a release mechanism activated by inositol triphosphate), and the mitochondrion (a voltage-dependent channel and an Na ⁺ /H ⁺ -dependent antiport system; see Fig. 13.34 ). The mechanisms for the increased cytosolic Ca ²⁺ in neurons subjected to hypoxia-ischemia, and the metabolic and ionic changes caused by ischemia that underlie these mechanisms, are summarized in Table 13.21 . The central roles for ATP depletion, membrane depolarization, and voltage-dependent and glutamate-activated Ca ²⁺ channels are apparent .

Cellular calcium (Ca ²⁺ ) homeostasis. See text for details. ADP , Adenosine diphosphate; AMPA , alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ATP ; adenosine triphosphate; DAG , diacylglycerol; ER , endoplasmic reticulum; Na ⁺ , sodium; NMDA , N -methyl- d -aspartate; PIP ₂ , phosphatidylinositol-4,5-diphosphate; PL-C , phospholipase C; VDCC , voltage-dependent calcium channel.

The deleterious effects of increased cytosolic Ca ²⁺ are multiple and affect the cell in a variety of ways ( Table 13.22 ). ^b

b References .

Deleterious effects of elevated cytosolic calcium ([Ca ⁺⁺ ]i).

Generation of free radicals is especially important. ATP , Adenosine triphosphate. See text for details.

Free Radicals.

The crucial role of free radicals, generated in considerable part by Ca ²⁺ -activated processes just described, in the mediation of cell death with hypoxia-ischemia has been established by the study of a variety of models in vivo, in culture, and in vitro (see later). The emphasis in these studies has been neuronal injury. Discussion of the role of free radicals in oligodendroglial injury is contained in a later section. Free radicals are highly reactive compounds with an uneven number of electrons in the outermost orbital. These compounds can react with certain normal cellular components (e.g., unsaturated fatty acids of membrane lipids) and can generate a new free radical and thereby a chain reaction, which results in irreversible biochemical injury (e.g., peroxidation of the unsaturated fatty acids, membrane injury, and cell necrosis). With less intense insults, free radicals can lead to apoptotic cell death by activation of specific death genes. Free radicals in mammalian cells exist primarily as ROS (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical) or as RNS (e.g., peroxynitrite [ONOO ⁻ ]; see later discussion). When these reactive species are present in excess, the cell is said to be subjected to oxidative stress or nitrative stress, respectively. Although studies have generally focused on ROS or RNS separately, these species usually exist together (e.g., under conditions of hypoxia-ischemia-reperfusion). I discuss the investigations of ROS and RNS in separate sections later.

The principal sources of free radicals with hypoxia-ischemia, the endogenous defenses against such radicals, and their major deleterious effects are summarized in Table 13.23 . Of the sources of free radicals with hypoxia-ischemia, the electron transport system is important when oxygen deprivation prevents the complete passage of electrons to cytochrome c oxidase. Free radicals, specifically superoxide anion, are then generated proximal to this terminal enzyme in the electron transport system. The next four sources are directly or indirectly related to cytosolic Ca ²⁺ (see Fig. 13.35 and Table 13.22 ). Arachidonic acid is generated by Ca ²⁺ -activated phospholipase A ₂ , xanthine oxidase is activated by Ca ²⁺ , catecholamine release is stimulated by an increase in cytosolic Ca ²⁺ , and NO synthase (NOS; see next section) is activated by Ca ²⁺ . ^a

a References .

Reactive Oxygen Species.

An important role for ROS in perinatal models of fetal and neonatal hypoxemic, ischemic, hypoxic-ischemic, and asphyxial insults now seems established. ^b

b References .

c References .

Free radical (superoxide anion) production, determined as superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction in control ( n = 7), asphyxia without reventilation ( n = 9), asphyxia-reventilation ( n = 11), asphyxia-reventilation after indomethacin 0.2 mg/kg ( n = 4), and asphyxia-reventilation after oxypurinol 50.0 mg/kg ( n = 10) pretreated piglets. Valuesare mean ± SEM. * P < .05 compared with control; ⁺⁺ P < .05 compared with asphyxia without reventilation group; ⁺⁺⁺ P < .05 compared with indomethacin and oxypurinol pretreatment groups. Note the production of superoxide anion after (with reventilation) but not during (without reventilation) asphyxia and the prevention by administration of indomethacin or oxypurinol.

(From Pourcyrous M, Leffler CW, Bada HS, Korones SB, Busija DW. Brain superoxide anion generation in asphyxiated piglets and the effect of indomethacin at therapeutic dose. Pediatr Res . 1993;34:366–369.)

The major endogenous antioxidant defense system is illustrated in Fig. 13.37 . Thus the most commonly generated initial oxygen free radical, the superoxide anion, is converted to hydrogen peroxide by the enzyme SOD, the three different forms of which are cytosolic copper-zinc SOD, extracellular copper-zinc SOD, and mitochondrial manganese SOD. The hydrogen peroxide generated is detoxified by catalase and glutathione peroxidase. If this step fails or is overloaded, and if iron is available, the Fenton reaction and the production of the deadly hydroxyl radical occur. Studies of animal models and more recent studies of asphyxiated human infants indicate that after hypoxia-ischemia, iron is released and is therefore relatively abundant. Studies of hypoxia-ischemia in the neonatal mouse indicate that detoxification of hydrogen peroxide is deficient in the immature brain. Thus mice made transgenic for copper-zinc SOD and therefore overexpressing this enzyme, when subjected to hypoxia-ischemia, exhibit decreased brain injury in the adult but increased brain injury in the perinatal period. This exacerbation of brain injury in the immature brain was associated with an accumulation of hydrogen peroxide, because catalase and glutathione peroxidase did not increase in activity in response to the insult (catalase increases after similar hypoxia-ischemia in the adult). Indeed, the levels of glutathione peroxidase decreased after hypoxia-ischemia, and importantly, in the normal animal levels already developmentally low in the perinatal period. Consistent with this, in normally developing rats (P7) rendered hypoxic-ischemic, an accumulation of hydrogen peroxide in the cortex was much greater than occurred with hypoxia-ischemia at later ages (P42). The particular importance of glutathione peroxidase was shown in two models derived from transgenic mice overexpressing glutathione peroxidase. Thus, in cultured neurons exposed to oxidative stress and in a neonatal hypoxic-ischemic model, glutathione peroxidase overexpression led to neuronal protection. Additional importance for glutathione is suggested by the finding in the hypoxic-ischemic 7-day rat that the crucial mitochondrial fraction of glutathione, necessary for glutathione peroxidase function, was markedly decreased after hypoxia-ischemia, with a nadir after 24 hours. Thus the data suggest that the normal immature brain has a limited capacity to detoxify hydrogen peroxide and with hypoxia-ischemia accumulates hydrogen peroxide, both because of this developmental lack and because of failure to respond with an adaptive increase in the antioxidant defense enzymes. With the hypoxia-ischemia–induced increase in iron, generation of the hydroxyl radical and brain injury is the result (see Fig. 13.37 ).

Free radical metabolism.

The upper two reactions, catalyzed respectively by superoxide dismutases (SOD; both copper-zinc SOD [extracellular and cytosol] and manganese SOD [mitochondrion]) and by GSH peroxidase and catalase, are the major antioxidant defense mechanisms. In the presence of hydrogen peroxide (H ₂ O ₂ ) and iron (Fe ⁺⁺ ), the Fenton reaction (lowest reaction) generates the highly toxic hydroxyl radical. GSH , Glutathione; SOD , superoxide dismutases.

The propensity for oxidative neuronal injury in the immature brain relates not only to the deficient antioxidant defenses just described but also to several pro-oxidant characteristics . Thus, at baseline, the developing brain has a relatively high concentration of polyunsaturated fatty acids, especially in neuronal membranes, an excellent target for ROS, and a relatively high concentration of nonprotein-bound iron. Therefore, with exposure to conditions that lead to an increase in ROS (e.g., hypoxia-ischemia-reperfusion) and available iron, the balance strongly favors oxidative injury. Similar considerations apply to the increased oxidative injury observed after hypoxia-ischemia in developing animals or in asphyxiated infants when resuscitation is carried out with 100% oxygen rather than with room air (see Chapter 20 ).

Nitric Oxide and Reactive Nitrogen Species.

Particular importance for the synthesis of NO by NOS both in normal brain and under conditions of hypoxia-ischemia is now well established. At least three forms of NOS are recognized: a constitutive neuronal form (nNOS), a constitutive endothelial form (eNOS), and an inducible form (iNOS) found in astrocytes and microglia. The constitutive forms are activated by Ca ²⁺ , whereas iNOS stimulation appears to be Ca ²⁺ independent and is activated especially well by inflammatory stimuli (e.g., cytokines, lipopolysaccharide [LPS], and oxidative stress). Because NO is a diffusible gas, both its normal functions (i.e., cell signaling and neurotransmission) and its deleterious actions (i.e., neurotoxicity) appear to be mediated by synthesis and then diffusion to intracellular sites and to adjacent cells.

Under normal conditions, NO has multiple cellular effects. Many of these, but not all, are initiated by guanylate cyclase activation with the subsequent production of cyclic guanosine monophosphate and protein phosphorylation. This activation occurs when NO binds to the heme group of guanylate cyclase. Other biological effects of NO relate to action of its metabolites. Thus formation of a nitro group (NO ₂ ) allows the nitration of proteins, especially at tyrosine residues, to form nitrotyrosine. Formation of nitrosonium ion (NO ⁺ ) allows nitrosylation of thiol residues on proteins, especially the cysteine sulfhydryl group. Formation of ONOO ⁻ and other reactive intermediates, including the hydroxyl radical, leads to oxidation of multiple amino acid residues, including the formation of nitrotyrosine. The biological effects of these actions of NO include modulation of cell proliferation, apoptosis, mitochondrial energy metabolism, and signal transduction.

Under pathological conditions of hypoxia-ischemia-reperfusion , an increase in cytosolic Ca ²⁺ is an early event (see earlier). A particularly important effector for the increase in Ca ²⁺ is activation of the NMDA receptor (see later). The effects on NO metabolism are multiple and sequential ( Fig. 13.38 ). Thus, in the first few minutes, the activity of Ca ²⁺ -dependent eNOS in endothelial cells is increased to produce vasodilation and to replenish substrate supply. The activity of Ca ²⁺ -dependent nNOS in neurons also is increased, and initially this increase may help improve blood flow because of the presence of nNOS in perivascular nerves. However, the principal effect of induction of nNOS in neurons is the generation of NO that diffuses to adjacent neurons. Under conditions of oxidative stress with abundant superoxide anion ( ), NO reacts quickly with to form the particularly toxic RNS, ONOO ⁻ . Indeed, the affinity of for NO to form ONOO ⁻ greatly exceeds the reaction of with SOD, and thus ONOO ⁻ formation is greatly favored. This compound leads to neuronal death by multiple mechanisms, including especially mitochondrial impairment , energy depletion, and further failure of Ca ²⁺ -homeostasis (see earlier). Glutamate activation of the NMDA receptor is a major cause of the initial increase of cytosolic Ca ²⁺ that activates nNOS and leads to this deleterious cascade. Those NMDA receptor–containing neurons that express nNOS are resistant to the deleterious effects of hypoxia-ischemia and excitotoxicity (see later).

Major effects of the various forms of nitric oxide synthase (NOS) after hypoxia-ischemia.

Effects in the first minutes to hours and in the hours to days after the insult can be distinguished, although these effects overlap. Dotted lines indicate neuroprotective effects that appear plausible under certain circumstances but require more study. See text for details. eNOS , Endothelial NOS; iNOS , inducible NOS; nNOS , neuronal NOS.

A later result of hypoxia-ischemia-reperfusion is the activation of iNOS , principally in astrocytes and microglia, with a resulting large, sustained increase in NO production, over many hours to days, depending on the experimental model studied (see Fig. 13.38 ). This activation occurs in parallel with inflammatory responses and is based on the activation of specific cell surface receptors for cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma, and interleukin-1beta [IL-1beta]) and on the intracellular effects of ROS. The deleterious effects of this increase in NO are mediated primarily through formation of ONOO ⁻ , as discussed earlier. The possibility of a neuroprotective role of NO, mediated by NO ⁺ , has been raised, because S -nitrosylation at critical thiols on the NMDA receptor’s redox modulatory site leads to downregulation of channel activity. The biological impact of such a neuroprotective role currently is unclear.

In perinatal models of asphyxia or hypoxia-ischemia , evidence for both the neurotoxic effects and the beneficial vascular effects of NO synthesis have been obtained. ^a

a References .

Normal Features.

The relationships at the glutamate synapse among the presynaptic nerve ending, the postsynaptic dendrite, and the associated astrocyte are shown in Fig. 13.39 . Only the ionotropic receptors are shown in Fig. 13.39 (as discussed in the next paragraph). Glutamate release is provoked by the influx of Ca ²⁺ into the presynaptic nerve ending. Depolarization of the postsynaptic dendrite is related to Na ⁺ entry. The action of glutamate is terminated by potent, high-affinity, Na ⁺ -dependent reuptake mechanisms in both astrocytes and presynaptic nerve endings. Although the transporters per se are not energy dependent, energy failure, as with hypoxia-ischemia, leads to disruption of Na ⁺ -K ⁺ ionic gradients across the plasma membrane because of failure of the ATP-dependent Na ⁺ -K ⁺ pump; the results are impairment of the Na ⁺ -dependent glutamate transporters and ultimately reversal of transporter function. In the astrocyte, the ATP-dependent enzyme, glutamine synthetase, uses ammonia and glutamate to form glutamine, which diffuses to the presynaptic nerve ending to regenerate glutamate on removal of this second amino group (see Fig. 13.39 ). ATP depletion also causes this mechanism to fail, and thus clearly ATP depletion results in failure of the major reuptake and removal mechanisms and leads to accumulation of extracellular glutamate and to excitotoxicity (see the following discussion).

Relationships at the glutamate synapse among the presynaptic axonal terminal, the postsynaptic dendrite, and the associated astrocyte.

See text for details. In this figure, the non- N -methyl- d -aspartate (NMDA; kainate-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid [KA-AMPA]) receptors are shown together; see text regarding calcium (Ca ⁺⁺ ) permeability of the AMPA receptor in developing neurons. ADP, Adenosine diphosphate; ATP , adenosine triphosphate; Cl ⁻ , chloride; Gln , glutamine; Glu , glutamate; Na ⁺ , sodium; NH ₃ , ammonia; VDCC , voltage-dependent calcium channel.

(Modified from Siesjö BK. Calcium in the brain under physiological and pathological conditions. Eur Neurol . 1990;30:3–9.)

These mechanisms of reuptake and removal must be highly efficient, because although the intracellular concentration of glutamate is extraordinarily high (i.e., 5 to 10 mmol/kg), the extracellular concentration is approximately 1000-fold less (i.e., in the low micromolar range or perhaps lower). The high concentration of glutamate in neurons implies a large release of glutamate into the extracellular space when cell death occurs, an occurrence that is relevant not only to amplification of primary excitotoxic cell death, as occurs with hypoxia-ischemia, but also to the development of secondary excitotoxic cell death from other types of injuries to neurons (e.g., trauma). In addition, release of glutamate from astrocytes may be even more marked than from neurons, under ischemic conditions.

Glutamate acts at both ionotropic and metabotropic receptors ( Table 13.24 ). Three of these are ionotropic (i.e., are linked to ion channels). The NMDA receptor is linked to an ion channel for Ca ²⁺ , the AMPA receptor is linked to a channel for Na ⁺ entry, and the kainate receptor is linked to a channel for Na ⁺ entry. The AMPA receptor is rendered Ca ²⁺ impermeable by the presence of one of its four subunits—namely, the GluR2 subunit. This subunit is relatively sparse during early development both in rodent and human neurons, and this feature renders the AMPA receptor in immature neurons Ca ²⁺ permeable. This feature may underlie the involvement of AMPA receptors in the hypoxic-ischemic or glutamate-induced death of immature neurons (see later discussion). Although the NMDA receptor often is considered the most crucial for the excitotoxic effects of glutamate in developing neurons, data suggest comparable importance for the AMPA receptor (see later). Agents that increase or decrease glutamate activation at the synapse mediated by the NMDA receptor-channel complex are shown in Table 13.25 . A fourth glutamate receptor type is metabotropic (i.e., is coupled through a guanosine triphosphate binding protein [G protein] to an enzyme producing a second messenger, phospholipase C, for phosphoinositide hydrolysis). The resulting products, diacylglycerol and inositol triphosphate, function as second messengers, the former activating protein kinase C, which has many cellular effects, and the latter promoting Ca ²⁺ mobilization from the endoplasmic reticulum (see Fig. 13.35 ).

The normal ontogeny of glutamate receptors is relevant to normal brain development and to the vulnerability of immature brain regions to excitotoxic cell death with hypoxia-ischemia. Detailed earlier studies of the development of binding sites for NMDA and non-NMDA receptor agonists in the rat showed a striking increase in the early phases of brain development. Later work, largely based on immunocytochemical and in situ hybridization studies, demonstrated the marked developmental increase more clearly, with peak values for the NMDA and AMPA receptors that exceed those in the adult brain. The peak values for NMDA and AMPA receptors were reached at only slightly different ages, with the NMDA peak preceding the AMPA peak. The timing of these peaks in the rat correlated with the perinatal period for the human brain . This can be depicted in Fig. 13.40 and is discussed with relevance to preterm brain injury ( Chapter 14 , Chapter 15 , Chapter 16 ) and term brain injury ( Chapter 18 , Chapter 19 , Chapter 20 ).

Schematic depiction of maturational changes in glutamate and GABA receptor function in the developing brain.

Equivalent developmental periods are displayed for rats and humans on the top and bottom axes, respectively. Activation of GABA receptors is depolarizing in rats early in the first postnatal week and in humans up to and including the neonatal period. Functional inhibition, however, is gradually reached over development in rats and humans. Before full maturation of GABA-mediated inhibition, the NMDA and AMPA subtypes of glutamate receptors peak between the first and second postnatal weeks in rats and in the neonatal period in humans. Kainate receptor binding is initially low and gradually rises to adult levels by the fourth postnatal week. AMPA , α-amino-3-hydroxy-5-methyl-4-isoxazole propionate; GABA , γ-aminobutyric acid; NMDA , N -methyl- d -aspartate; P , postnatal day.

(Reprinted with permission from Rakhade SN, Jensen FE. Epileptogenesis in the immature brain: emerging mechanisms. Nat Rev Neurol . 2009;5:380–391.)

The molecular characteristics of the NMDA and AMPA receptors also are developmentally regulated, and the major characteristics of the developing receptors indicate enhanced Ca ²⁺ influx. Thus, regarding the NMDA receptor, the relative expression of the NR2B subunit compared with that of the NR2A subunit is increased in the immature brain versus that of the adult. NMDA receptors containing predominantly NR2B exhibit increased duration of NMDA receptor–mediated excitation and increased Ca ²⁺ influx . With regard to the AMPA receptor, in the immature brain the expression of the GluR2 subunit , which renders the AMPA receptor Ca ²⁺ impermeable, as in the mature brain, is relatively low . Thus the large numbers of AMPA receptors in developing neurons are largely permeable to Ca ²⁺ .

The transient, dense expressions of these ionotropic glutamate receptors of enhanced functional capabilities have implications not only for their role in normal development but also in determining neuronal death with hypoxia-ischemia (see later). The role of glutamate receptor activation and Ca ²⁺ influx relates to such processes as regulation of neurite outgrowth, synapse formation, cell death, selective elimination of neuronal processes, and functional organization of neuronal systems. However, in addition, these transient dense expressions of glutamate receptors of enhanced functional capabilities may become the unintended mediators of neuronal death with hypoxia-ischemia (see later discussion). Moreover, the likelihood that these principles apply to the developing human brain is supported by the demonstration of early overexpression of glutamate receptors in human hippocampus, cerebral cortex, deep nuclear structures (i.e., basal ganglia and thalamus), and certain brain stem nuclei—regions vulnerable to hypoxic-ischemic injury in the newborn (see Chapter 14 , Chapter 15 , Chapter 16 , Chapter 17 , Chapter 18 , Chapter 19 , Chapter 20 ).

Role of Glutamate in Hypoxic-Ischemic Cell Death in Cultured Neurons.

The critical role for glutamate in the mediation of hypoxic-ischemic neuronal death is established by a large body of experimental information, as summarized in Table 13.26 . ^a

a References .

The crucial initial observation was that cultured hippocampal neurons, obtained from the fetal rat, were resistant to prolonged anoxia before synapse formation occurred in the cultures, but they were very sensitive to the same anoxic insult after synaptogenesis was well developed. Thus, such mature cultures markedly deteriorated in the absence of oxygen. However, when synaptic activity in these mature cultures was blocked by the addition of high concentrations of magnesium, no effect of anoxia on the cultured neurons occurred ( Fig. 13.41 ). Thus the data demonstrated that synaptic activity resulted in neuronal death with oxygen deprivation. This protection by synaptic blockage with magnesium was shown later in a hippocampal slice preparation in which neuronal death could be produced in the CA1 region under anoxic conditions.

Effect of blockade of synaptic activity on anoxic cell death in hippocampal neuronal cultures.

(A) Phase contrast micrograph ( top ) shows normoxic culture with abundant neurons; bottom , cultures rendered anoxic for 24 hours show extensive neuronal destruction provoked by anoxia. (B) Micrograph shows cultures treated with magnesium chloride to block synaptic activity before (top) and after (bottom) anoxia. Note the lack of neuronal destruction.

(From Rothman SM. Synaptic activity mediates death of hypoxic neurons. Science. 1983;220:536–537.)

Because glutamate (as in hippocampus in vivo) was presumed to be the neurotransmitter mediating the synaptic activity in the experiments with the cultured hippocampal neurons and the slice preparation, a nonspecific postsynaptic blocker of glutamate was investigated to prevent the hypoxic neuronal death in culture. This agent protected neurons dramatically from anoxia ( Fig. 13.42 ). The particular role of glutamate synapses in hippocampal neuronal death was supported further shortly thereafter by the demonstration that hypoxic-ischemic neuronal injury could be prevented in vivo by prior section of glutamatergic afferents to the CA1 region.

Effect of a blocker of the N -methyl- d -aspartate (NMDA)-type glutamate receptor on cell death in hippocampal neuronal cultures.

(A) Phase contrast micrographs show cultures before (top) and 8 hours after (bottom) anoxia. Note neuronal destruction after anoxia. (B) Micrographs show cultures treated with the NMDA receptor blocker, gamma- d -glutamylglycine. The appearance before (top) and 8 hours after (bottom) anoxia are shown. Note the prevention of neuronal destruction in the presence of the blocker.

(From Rothman SM. Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J Neurosci . 1984;4:1884.)

The mechanisms of glutamate-induced neuronal death in cultured neurons were elucidated next. ^a

a References .

b References .

Mechanisms of glutamate-induced neuronal death.

Note the involvement of the N -methyl- d -aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, the increase in intracellular calcium ([Ca ²⁺ ] _i ) the generation of reactive oxygen (ROS) and reactive nitrogen (RNS) species, and the resulting cell death.

Relevance of Glutamate-Induced Excitotoxicity to Hypoxic-Ischemic Injury in Vivo.

The relevance of the glutamate excitotoxic mechanisms to the in vivo situation is now clear. ^a

a References .

b References .

The second body of evidence delineating the relevance of glutamate to the in vivo situation is the demonstration in a wide variety of perinatal models of hypoxia-ischemia that glutamate is toxic to neurons in vivo and that this toxicity is particularly marked in the immature versus the mature animal. ^c

c References .

d References .

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