Delayed cerebral atrophy following moderate hypoxia-ischemia in the immature rat.

Hypoxia-ischemia (H/I) damages cells in the immature brain and interferes with subsequent brain development; the extent of the damage has been related to the severity, or duration, of the initial insult. This study examined the effects of both severe and moderate duration of H/I on the evolution of damage through 8 weeks of recovery. Seven-day-old rat pups were subjected to either 75 min or 2 h of 8% oxygen following a unilateral carotid artery ligation. Evaluation of brain damage included morphometric analysis of hemispheric diameter at 2, 4, and 8 weeks of recovery, and hematoxylin and eosin for evaluation of pathology at 8 weeks. Two hours of H/I produced severe infarction in the ipsilateral hemisphere in the majority of the survivors, apparent by 2 weeks of recovery with no change at 4 or 8 weeks. In marked contrast, 75 min of H/I produced no significant damage during the initial 2 weeks of recovery but resulted in progressive cerebral atrophy with delayed infarction such that the extent of damage at 8 weeks was not different from the 2-hour group. Thus, even a mild-moderate ischemic insult to the perinatal brain establishes a vulnerable region which ultimately dies without intervention.

Hypoglycemic brain injury.

Hypoglycemia frequently occurs in newborn infants who previously have suffered asphyxia, who are offspring of diabetic mothers, or who are low birthweight for gestational age (IUGR). Many infants who are hypoglycemic do not exhibit clinical manifestations, while others are symptomatic and at risk for the occurrence of permanent brain damage. This review emphasizes the clinical, neuropathologic, and neuro-imaging features of hypoglycemia in newborn infants, especially those who are symptomatic. Neurologic morbidity occurs particularly in those infants who have suffered severe, protracted, or recurrent symptomatic hypoglycemia. Experimental observations emphasize the resistance of the immature brain to the damaging effect of hypoglycemia; such resistance occurs as a consequence of compensatory increases in cerebral blood flow, lower energy requirements, higher endogenous carbohydrate stores, and an ability to incorporate and consume alternative organic substrates to spare glucose for energy production. Hypoglycemia combined with hypoxia-ischemia (asphyxia) is more deleterious to the immature brain than either condition alone.

Effect of extreme hypercapnia on hypoxic-ischemic brain damage in the immature rat.

To ascertain the effect of extreme hypercapnia on perinatal hypoxic-ischemic brain damage, 7-d-postnatal rats were exposed to unilateral common carotid artery occlusion followed by hypoxia with 8% oxygen combined with 3, 12, or 15% carbon dioxide (CO2) for 2 h at 37 degrees C. Survivors underwent neuropathologic examination at 30 d of postnatal age, and their brains were characterized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = cystic infarct with external dimensions <3 mm; and 4 = cystic infarct with external dimensions >3 mm. The width of the cerebral hemisphere ipsilateral to the carotid artery occlusion also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. CO2 tensions averaged 5.08, 11.1, and 13.2 kPa in the 3, 12, and 15% CO2-exposed animals, respectively, during hypoxia-ischemia (HI). Neuropathologic results showed that immature rats exposed to 3 and 12% CO2 had similar severities of brain damage. In contrast, rat pups exposed to HI combined with 15% CO2 were significantly more brain damaged than littermates exposed to 3% CO2. Specifically, eight of 14 animals exposed to 15% CO2 showed cystic infarcts (grades 3 and 4), whereas none of 14 littermates exposed to 3% CO2 developed cystic infarcts (p < 0.01). Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. Cerebral blood flow (CBF), measured at 90 min of HI, was lowest in those immature rats exposed to 15% CO2 compared with control (p = 0.04), with higher values in those rat pups exposed to 3 and 12% CO2. The findings indicate that 7-d-postnatal rats exposed to HI with superimposed 12% CO2 are neither less nor more brain damaged than littermates exposed to 3% CO(2) (normocapnia). In contrast, animals exposed to 15% CO2 are the most brain damaged of the three groups. Presumably, extreme hypercapnia produces more severe cardiovascular depression than is seen in animals subjected to lesser degrees of hypercapnia; the cardiovascular depression, in turn, leads to greater cerebral ischemia and ultimate brain damage.

Is periventricular leucomalacia a result of hypoxic-ischaemic injury? Hypocapnia and the preterm brain.

Decrease in the arterial partial pressure of carbon dioxide (PaCO(2)) causes a reduction in cerebral blood flow in humans and in most animal species; in adults as well as in newborns and even in fetal life. Severely decreased PaCO(2) increases cerebral lactate production, modifies spontaneous electric brain activity, and may decrease the metabolic rate of oxygen. A relation between very low PaCO(2) and brain injury, however, has not been shown in adult humans or full-term newborn infants, nor in perinatal animals. In contrast, an association between low PaCO(2) and cerebral palsy and white matter injury in preterm infants has been reported repeatedly. A cause-and-effect relation is suggested by data from the immature rat: brain damage induced by ligation of a carotid artery can be reduced by adding CO(2) to the inspired gas and hence avoiding the consequences of spontaneous hyperventilation. This may be relevant for the clinical care of preterm infants, since PaCO(2) to a large extent is a function of respiratory management. The questions to be addressed are whether hypocapnia sensitizes the brain to hypoxaemia, and also whether the escape mechanisms are less effective in the preterm human brain.

Intracellular calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat.

An excessive intracellular accumulation of calcium (Ca2+) in neurons and glia has been proposed to represent a major 'final common pathway' for cell death arising from hypoxia-ischemia. To clarify the role of altered calcium flux into the perinatal brain undergoing hypoxic-ischemic damage, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by systemic hypoxia with 8% oxygen. This insult is known to produce brain damage in the form of selective neuronal death or infarction largely limited to the cerebral hemisphere ipsilateral to the arterial occlusion. Either prior to or following hypoxia-ischemia, the rat pups received a s.c. injection of 45CaCl2, and specimens of blood, cerebrospinal fluid (CSF), and brain were obtained for isotopic measurements and the calculation of the extent of brain intracellular radioactivity. During hypoxia-ischemia, there was a modest increase in intracellular Ca2+ radioactivity (+28-47%) in both cerebral hemispheres only after 2 h of hypoxia-ischemia. During recovery from 2 h of hypoxia-ischemia, intracellular Ca2+ accumulated progressively only in the ipsilateral cerebral hemisphere for up to 24 h, during which interval intracellular Ca2+ decreased in the contralateral hemisphere. No such progressive accumulation was noted during recovery in animals previously exposed to only 1 h of hypoxia-ischemia. The results suggest that a disruption of intracellular Ca2+ homeostasis is a major contributing factor in the evolution of perinatal hypoxic-ischemic brain damage. Ca2+ accumulation is a relatively modest and late event during the hypoxic-ischemic phase, and a progressive overload occurs during the recovery phase only if infarction occurs. The question remains as to whether or not the intracellular Ca2+ overload occurring during recovery is a contributor to or a consequence of the ultimate brain damage.

Hypoxic-ischemic encephalopathy.

Cerebral hypoxia-ischemia (asphyxia) occurring in the fetus and newborn infant is a major cause of acute mortality and chronic neurological disability in survivors. This review highlights many practical aspects of perinatal hypoxic-ischemic brain damage, including neuropathological features, obstetrical antecedents, and clinically important aspects of identification, management, and prognosis. Diagnostic techniques, including neuro-imaging, to diagnose hypoxic-ischemic encephalopathy also are discussed. A thorough knowledge of the clinical spectrum of perinatal hypoxic-ischemic encephalopathy should enable neonatologists to undertake appropriate management strategies and prognostic indicators.

Increased plasma beta-hydroxybutyrate, preserved cerebral energy metabolism, and amelioration of brain damage during neonatal hypoxia ischemia with dexamethasone pretreatment.

Dexamethasone (DEX) pretreatment has been shown to be neuroprotective in a neonatal rat model of hypoxia ischemia (HI). The exact mechanism of this neuroprotection is still unknown. This study used 31P nuclear magnetic resonance spectroscopy to monitor energy metabolism during a 3-h episode of HI in 7-d-old rat pups in one of two groups. The first group was pretreated with 0.1 mL saline (i.p.) and the second group was treated with 0.1 mL of 0.1mg/kg DEX (i.p.) 22 h before HI. Animals pretreated with DEX had elevated nucleoside triphosphate and phosphocreatine levels during HI when compared with controls. Saline-treated animals had significant decreases in nucleoside triphosphate and phosphocreatine and increases in inorganic phosphate over this same period. 31P nuclear magnetic resonance data unequivocally demonstrate preservation of energy metabolism during HI in neonatal rats pretreated with DEX. Animals pretreated with DEX had little or no brain damage following 3 h of HI when compared with matched controls, which experienced severe neuronal loss and cortical infarction. These same pretreated animals had an increase in blood beta-hydroxybutyrate levels before ischemia, suggesting an increase in ketone bodies, which is the neonate's primary energy source. Elevation of ketone bodies appears to be one of the mechanisms by which DEX pretreatment provides neuroprotection during HI in the neonatal rat.

Glucose metabolism in the developing brain.

As in adults, glucose is the predominant cerebral energy fuel for the fetus and newborn. Studies in experimental animals and humans indicate that cerebral glucose utilization initially is low and increases with maturation with increasing regional heterogeneity. The increases in cerebral glucose utilization with advancing age occurs as a consequence of increasing functional activity and cerebral energy demands. The levels of expression of the 2 primary facilitative glucose transporter proteins in brain, GLUT1 (blood-brain barrier and glia) and GLUT3 (neuronal), display a similar maturational pattern. Alternate cerebral energy fuels, specifically the ketone bodies and lactate, can substitute for glucose, especially during hypoglycemia, thereby protecting the immature brain from potential untoward effects of hypoglycemia. Unlike adults, glucose supplementation during hypoxia-ischemia is protective in the immature brain, whereas hypoglycemia is deleterious. Accordingly, glucose plays a critical role in the developing brain, not only as the primary substrate for energy production but also to allow for normal biosynthetic processes to proceed.

CSF glutamate during hypoxia-ischemia in the immature rat.

Cerebrospinal fluid (CSF) glutamate was measured prior to and during the course of cerebral hypoxia-ischemia in the immature rat to estimate its concentration in the extracellular fluid (ECF). A preliminary experiment was conducted using [14C]glutamate injections into immature rat brain, which showed that equilibration between ECF and CSF occurred within 10 min. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed by hypoxia with 8% oxygen for up to 2 h. Brain damage, in the form of selective neuronal necrosis or apoptosis, commences after 60 min, while infarction commences after 90 min of hypoxia-ischemia. During the course of hypoxia-ischemia, CSF was obtained from the cisterna magna and analyzed for glutamate. No statistically significant increases in CSF glutamate occurred until 105 min, at which time the concentration was 240% of control (20 micromol/l). By 120 min, CSF glutamate had increased over twofold above the control value. In rat pups exposed to 1 h of hypoxia-ischemia, no increases in CSF glutamate occurred for up to 6 h of recovery. In animals exposed to 2 h of hypoxia-ischemia, CSF glutamate decreased to the control value by 1 h of recovery, with a secondary rise at 6 h. Accordingly, the increase in CSF, and presumably ECF, glutamate is a late event, which better corresponds temporally to cerebral infarction than to selective neuronal death. The results suggest that glutamate excitotoxicity, although involved in the occurrence of infarction, neither causes or contributes to selected neuronal death. The secondary elevation in CSF glutamate at 6 h of recovery from 2 h of hypoxia-ischemia occurs coincident with the onset of tissue necrosis, seen histologically.

Experimental models of hypothermic circulatory arrest.

This article reviews information obtained from experimental models of hypothermic circulatory arrest, which models have been developed in our and other laboratories over the past several years. The described experiments clearly demonstrate an ability to produce and completely reverse hypothermic circulatory arrest in newborn and developing animals, allowing for a comprehensive evaluation of those physiological variables and therapeutic interventions that would potentially reduce or accentuate ischemic brain damage. Further experiments will allow for a determination of whether or not specific modalities of therapy will reverse secondary systemic complications, thereby allowing for more complete recoverability and ultimately reduced brain damage.

Effect of seizures on cerebral hypoxic-ischemic lesions in immature rats.

The present investigation was designed to study the effect of chemically induced seizures on cerebral hypoxic-ischemic (HI) damage in immature animals. Accordingly, cerebral HI was produced in 7-day postnatal (p7) rats and p13 rats by combined unilateral common carotid artery ligation and hypoxia with 8% oxygen. Seizures were induced chemically by the subcutaneous injection of kainic acid (KA) or inhalation of flurothyl vapor. Three types of experiments were conducted in each age group and for each convulsant. In some animals (group 1), seizures were produced at 24 h and again at 6 h prior to HI. In groups 2 and 3, seizures were induced 2 h or 24 h post HI, respectively. The results indicate that in group 1 animals, the first seizure significantly reduced duration of the second seizure challenge 18 h later at both p7 and p13 (p=0.001). Histologic examination of brains of animals in group 1 subjected to seizures prior to HI and their HI-only controls showed that seizures prior to HI conferred protection against cerebral damage. This effect was significant for flurothyl seizures in p13 rats for all cerebral regions, especially hippocampal CA1 (p=0.0004), and in p7 rats for hippocampus (p=0.04) and particularly cerebral cortex (p=0.007). For KA seizures, the protective effect was only significant in p13 rats and was limited to hippocampal CA regions and subiculum (p=0.0009). Histologic assessment of cerebral lesions of p7 and p13 rats in the other two groups showed no significant difference between the animals subjected to seizures 2 h or 24 h post HI and their HI-only controls (p>0.05). In conclusion, the results of the present study provide no evidence that seizures in early postnatal development aggravate pre-existing cerebral HI damage. They do suggest that seizures prior to HI or prior to a second seizure confer tolerance to both conditions.

Rat model of perinatal hypoxic-ischemic brain damage.

To gain insights into the pathogenesis and management of perinatal hypoxic-ischemic brain damage, the authors have used an immature rat model which they developed many years ago. The model entails ligation of one common carotid artery followed thereafter by systemic hypoxia. The insult produces permanent hypoxic-ischemic brain damage limited to the cerebral hemisphere ipsilateral to the carotid artery occlusion. The mini-review describes recently accomplished research pertaining to the use of the immature rat model, specifically, investigations involving energy metabolism, glucose transporter proteins, free radical injury, and seizures superimposed upon cerebral hypoxia-ischemia. Future research will focus on molecular mechanisms of neuronal injury with a continuing focus on therapeutic strategies to prevent or minimize hypoxic-ischemic brain damage.

Hypoxic preconditioning and hypoxic-ischemic brain damage in the immature rat: pathologic and metabolic correlates.

It has been reported that immature rats subjected to cerebral hypoxia-ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic-ischemic brain damage. Six-day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37 degrees C for 2.5 h. Twenty-four hours later, they were exposed to unilateral cerebral hypoxia-ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction (p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high-energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia-ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high-energy stores during the first 24 h of recovery from hypoxia-ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high-energy reserves during hypoxia-ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia-ischemia.

Alterations in GLUT1 and GLUT3 glucose transporter gene expression following unilateral hypoxia-ischemia in the immature rat brain.

The brain damage produced by unilateral cerebral hypoxia-ischemia in the immature rat results from major alterations in cerebral energy metabolism and glucose utilization which begin during the course of the insult and proceed into the recovery period. Consistent with a lack of pathology, the alterations in the hemisphere contralateral to the carotid artery ligation are transient and return to normal within 24 h of recovery, whereas the hemisphere ipsilateral to the ligation exhibits both early and late responses, and infarction. The facilitative glucose transporter proteins mediate glucose transport across the blood-brain barrier (55 kDa GLUT1), and into neurons and glia (GLUT3 and 45 kDa GLUT1), and demonstrate both early and late responses to perinatal hypoxia-ischemia. This study employed in situ hybridization histochemistry to investigate the temporal and regional patterns of GLUT1 and GLUT3 gene expression following a severe (2.5 h) hypoxic-ischemic insult in the 7-day old rat brain. Enhanced GLUT1 mRNA expression was apparent in cerebral microvessels of both hemispheres and remained elevated in the ipsilateral hemisphere through 24 h of recovery, consistent with our previous observation of increased microvascular 55 kDa GLUT1 protein. The expression of the neuronal isoform, GLUT3, was enhanced in penumbral regions, such as piriform cortex and amygdala, but was rapidly reduced in the affected areas of cortex, hippocampus and thalamus, reflecting necrosis. The late response, observed at 72 h of recovery, was characterized by extensive necrosis in the ipsilateral hemisphere, loss of GLUT3 expression, and a gliotic reaction including increased GLUT1 in GFAP-positive astrocytes. This study demonstrates that cerebral hypoxia-ischemia in the immature rat produces both immediate-early and long-term effects on the glucose transporter proteins at the level of gene expression.

Effect of carbon dioxide on cerebral metabolism during hypoxia-ischemia in the immature rat.

We previously have demonstrated that hypocapnia aggravates and hypercapnia protects the immature rat from hypoxic-ischemic brain damage. To ascertain cerebral blood flow (CBF) and metabolic correlates, 7-d postnatal rats were subjected to hypoxia-ischemia during which they were rendered either hypo-(3.5 kPa), normo- (5.1 kPa), or hypercapnic (7.3 kPa) by the inhalation of either 0, 3, or 6% CO2, 8% O2, balance N2. CBF during hypoxia-ischemia was better preserved in the normo- and hypercapnic rat pups; these animals also exhibited a stimulation of cerebral glucose utilization. Brain glucose concentrations were higher and lactate lower in the normo- and hypercapnic animals, indicating that glucose was consumed oxidatively in these groups rather than by anaerobic glycolysis, as apparently occurred in the hypocapnic animals. ATP and phosphocreatine were better preserved in the normo- and hypercapnic rats compared with the hypocapnic animals. Cerebrospinal fluid glutamate, as a reflection of the brain extracellular fluid concentration, was lowest in the hypercapnic rats at 2 h of hypoxia-ischemia. The data indicate that during hypoxia-ischemia in the immature rat, CBF is better preserved during normo- and hypercapnia; the greater oxygen delivery promotes cerebral glucose utilization and oxidative metabolism for optimal maintenance of tissue high energy phosphate reserves. An inhibition of glutamate secretion into the synaptic cleft and its attenuation of N-methyl-D-aspartate receptor activation would further protect the hypercapnic animal from hypoxic-ischemic brain damage.

Influence of age on the cerebral lesions in an immature rat model of cerebral hypoxia-ischemia: a light microscopic study.

The most frequently used model of neonatal cerebral hypoxia-ischemia consists of a 7-day postnatal rat model with combined common carotid artery ligation and hypoxemia. Neuropathologic studies have shown major differences between this 7-day postnatal rat model and a similar adult model in regard to overall cerebral vulnerability, type and distribution of lesions. It is not clear how and when during animals' development these changes in cerebral vulnerability take place. To determine this we studied groups of rats of 2 to 30 postnatal days. The animals underwent unilateral common carotid artery ligation followed by breathing in 8% oxygen for 30, 60, 90, or 120 min and their brains were examined at 24- or 72-h recovery intervals. Due to resistance of 2-3-day-old rats to develop cerebral hypoxic-ischemic damage, 5% O2 was used instead of 8% O2. The results indicate that: (i) There is an overall increase in severity of cerebral lesions on the side of common carotid artery ligation between 2 and 7 postnatal days. There is also an increase in the frequency of cerebral lesions in developing animals with increasing age. (ii) Hippocampus is remarkably resistant to hypoxic-ischemic insult at 2-3 postnatal days but becomes progressively vulnerable, and by age 13 postnatal days hippocampal vulnerability far exceeds that of cortex. (iii) Cortical lesions change from predominantly columnar cell death to laminar selective neuronal death at age 13 postnatal days. (iv) Also significant changes occur in relative vulnerability of various hippocampal regions during development. During the first 5 postnatal days relative vulnerability of hippocampal regions is similar, but as the animals' development proceeds and hippocampal vulnerability increases lesions tend to involve specific regions while sparing others. By age 13 postnatal days CA1 and lateral CA3 develop increased vulnerability while medial CA3 and fascia dentata become relatively resistant and by 21 postnatal days adult pattern of CA1 selective vulnerability is approached. The underlying mechanisms for these changes in regional vulnerability to cerebral hypoxia-ischemia during development should be sought in complex regional anatomic, functional, and metabolic alterations that take place as brain matures.

Neuropathology of normothermic circulatory arrest in newborn dogs.

Neuropathologic findings are described, for the first time, in a neonatal dog model of circulatory arrest in normothermic conditions, and the findings are compared to those reported in neonatal dogs with hypothermic circulatory arrest. Total circulatory arrest was produced in 3- to 6-day-old anesthetized, paralyzed and ventilated, normothermic dogs either by asphyxiation or cardioplegia. Duration of circulatory arrest was 8-20 min and 10-40 min in asphyxiated and cardioplegic animals, respectively. The animals were resuscitated and maintained under controlled systemic physiologic conditions until neuropathologic examination after 8 or 24 h of recovery. The results suggest that the minimal durations of circulatory arrest for brain damage to occur following asphyxia or cardioplegia are 10 and 15 min, respectively. Ischemic lesions in both groups consisted of neuronal necrosis and involved mainly the brain stem structures, particularly the reticular nuclei and the spinal cord gray matter. The medulla was more severely involved than midbrain and pons. There was a direct correlation between the length of circulatory arrest and the severity of damage in the medulla (P = 0.001) and overall brain stem damage (P = 0.004) in animals with cardioplegia, but not in animals with asphyxia. These findings are compared to the neuropathologic changes previously described in newborn dogs subjected to hypothermic circulatory arrest, in which ischemic lesions are focused on the cerebral cortex and basal ganglia. It is concluded that hypothermia in this model not only prolongs the period of circulatory arrest that is required to produce brain damage, but also shifts the pattern of regional ischemic vulnerability from caudal to more rostral structures.

Measuring the accentuation of the brain damage that arises from perinatal cerebral hypoxia-ischemia.

To ascertain the manner in which the severity of perinatal brain damage occurs as a result of hypoxia-ischemia, 7-day postnatal rats were subjected to unilateral common carotid artery ligation followed thereafter by exposure to 8% oxygen for up to 2.5 h. Following the hypoxic-ischemic exposure, the rat pups were reared with their dams until 30 days of postnatal age, at which time their brains underwent pathologic analysis. The severity of brain damage at each of four specific intervals of hypoxia-ischemia was determined and statistically compared by linear polynomial and nonparametric regression procedures. The data indicated that the accentuation of brain damage with increasing duration of hypoxia-ischemia was linear rather than curvilinear.

A model of perinatal hypoxic-ischemic brain damage.

In conclusion, our immature rat model has gained wide acceptance as the animal model of choice to study basic physiologic, biochemical, and molecular mechanisms of perinatal hypoxic-ischemic brain damage. In addition, the model has been used extensively to study those physiologic and therapeutic variables which either are deleterious or beneficial to the perinatal brain undergoing hypoxia-ischemia. As therapeutic interventions are tested in the animal setting, the results will provide important information regarding the effect of these agents in the human setting.