Prolonged suppression of brain nitric oxide synthase activity by 7-nitroindazole protects against cerebral hypoxic-ischemic injury in neonatal rat.

Nitric oxide mediates glutamate-induced excitotoxicity associated with cerebral hypoxia-ischemia through production in the brain by several isoforms of nitric oxide synthase (NOS). We examined the influence of the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI), on brain NOS activity and its neuroprotective effects against cerebral hypoxic-ischemic injury in the postnatal day (PND) 7 rat. In the first set of experiments, 7-NI (50 mg/kg) administered intraperitoneally (i.p.) transiently inhibited NOS activity to 40% below the vehicle control level at 1 h after injection (P<0.001, analysis of variance (ANOVA)). In contrast, 7-NI (100 mg/kg, i.p.) inhibited NOS activity to 56% below the control level at 1 h with prolonged suppression of NOS activity at 3, 6, 9 and 12 h after injection. Two-factor ANOVA revealed an overall effect on NOS activity of 7-NI treatment (P<0.001) and time after injection (P<0.001). In the second set of experiments, 7-NI (50, 100 mg/kg) or an equal volume of vehicle was administered after unilateral carotid artery ligation, but 30 min before hypoxia in PND 7 rats. 7-NI (100 mg/kg) significantly protected against cerebral hypoxic-ischemic injury (100 mg/kg of 7-NI, 1.7+/-1.0% damage; control, 8.7+/-1.6%,P<0.05). 7-NI administered 15 min after cerebral hypoxia-ischemia was not neuroprotective. The data suggest that the protective effect of 7-NI is dose dependent, and is related to the duration of suppressed NOS activity.

Neurobiology of hypoxic-ischemic injury in the developing brain.

Hypoxic ischemia is a common cause of damage to the fetal and neonatal brain. Although systemic and cerebrovascular physiologic factors play an important role in the initial phases of hypoxic-ischemic injuries, the intrinsic vulnerability of specific cell types and systems in the developing brain may be more important in determining the final pattern of damage and functional disability. Excitotoxicity, a term applied to the death of neurons and certain other cells caused by overstimulation of excitatory, mainly glutamate, neurotransmitter receptors, plays a critical role in these processes. Selected neuronal circuits as well as certain populations of glia such as immature periventricular oligodendroglia may die from excitotoxicity triggered by hypoxic ischemia. These patterns of neuropathologic vulnerability are associated with clinical syndromes of neurologic disability such as the extrapyramidal and spastic diplegia forms of cerebral palsy. The cascade of biochemical and histopathologic events triggered by hypoxic ischemia can extend for days to weeks after the insult is triggered, creating the potential for therapeutic interventions.

Delayed increase in neuronal nitric oxide synthase immunoreactivity in thalamus and other brain regions after hypoxic-ischemic injury in neonatal rats.

We examined the response of neuronal nitric oxide synthase (nNOS)-containing CNS neurons in rats exposed to a unilateral hypoxic-ischemic insult at 7 days of age. Animals were sacrificed at several time points after the injury, up to and including 7 days (Postnatal Day 14). Brain regions ipsilateral to the injury (including cerebral cortex, caudate-putamen, and thalamus) exhibited delayed, focal increases in nNOS immunoreactivity. The increase in nNOS immunoreactive fiber staining was prominent in areas adjacent to severe neuronal damage, especially in the cortex and the thalamus, regions that are also heavily and focally injured in term human neonates with hypoxic-ischemic encephalopathy. In cerebral cortex, these increases occurred despite modest declines in nNOS catalytic activity and protein levels. Proliferation of surviving nNOS immunoreactive fibers highlights regions of selective vulnerability to hypoxic-ischemic insult in the neonatal brain and may also contribute to plasticity of neuronal circuitry during recovery.

Novel treatments after experimental brain injury.

Perinatal hypoxic-ischaemic encephalopathy(HIE) is being studied in laboratory models that allow the delayed cascade of events triggered by the energetic insult to be examined in detail. The concept of the 'excitotoxic cascade' provides a conceptual framework for thinking about the pathogenesis of HIE. Major events in the cascade triggered by hypoxia-ischaemia include overstimulation of N-methyl-D-aspartate type glutamate receptors, calcium entry into cells, activation of calcium-sensitive enzymes such as nitric oxide synthase, production of oxygen free radicals, injury to mitochondria, leading in turn to necrosis or apoptosis. New experimental approaches to salvaging brain tissue from the effects of HIE include inhibition of neuronal nitric oxide synthase, administration of neuronal growth factors, and inhibition of the caspase enzymes that execute apoptosis. Recent experimental work suggests that these approaches may be effective during a longer 'therapeutic window' after the insult, because they are acting on events that are relatively delayed. Application of modest hypothermia may allow these agents to be neuroprotective at even longer intervals after hypoxia-ischaemia.

Human FGF-1 gene delivery protects against quinolinate-induced striatal and hippocampal injury in neonatal rats.

Fibroblast growth factors (FGFs) are cell mitogens and differentiating factors with neuroprotective properties in the CNS. We have already shown that endothelial cells genetically engineered to secrete human FGF-1 (RBEZ-FGF) survive implantation to neonatal rat brain (Johnston et al. (1996) J. Neurochem. 67, 1643-1652]. In this study, the effects of cell-based FGF-1 gene delivery on quinolinate-induced neurotoxicity in the developing rat brain were examined. Control endothelial cells (RBE4), and RBEZ-FGF cells were implanted into right striatum at post-natal day (PND) 7. On PND 10, quinolinate (150 nmol), an endogenous N-methyl-d-aspartate (NMDA) receptor agonist, or vehicle alone was injected into striatum ipsilateral to cell implantation. Injury was quantified in coronal sections obtained from PND 17 animals by comparing striatal and hippocampal volumes ipsilateral and contralateral to the site of quinolinate injection. Human FGF-1 specific transgene expression in vivo was shown by Northern blot and RT-PCR up to 14 days after cell implantation in control animals, and up to 4 days after quinolinate exposure. Quinolinate reduced the size of ipsilateral striatum by 37% and hippocampus by 38% in animals preimplanted with control endothelial cells. In contrast, quinolinate reduced the size of striatum by only 14% and had no effect on hippocampal size in animals preimplanted with RBEZ-FGF cells. Thus, FGF-1 gene delivery protected the developing striatum and hippocampus from quinolinate-induced volume loss by 62% and 100%, respectively. Intrastriatal quinolinate resulted in a significant decrease in density of NOS+ CA3 hippocampal neurons (-38%) without affecting the density of NOS+ neurons in hippocampal regions CA1, dentate gyrus or striatum. This response of CA3 NOS+ neurons appeared to be only partially reversed by FGF-1 gene delivery. Our results show that intracerebral FGF-1 gene expression within the developing brain can protect striatum and hippocampus from quinolinate-mediated injury.

Brief post-hypoxic-ischemic hypothermia markedly delays neonatal brain injury.

The influence of post-insult temperature modulation on ischemic injury in immature brain was studied in 7-day-old rats that underwent a unilateral carotid artery ligation followed by exposure to hypoxia in 8% oxygen at an ambient temperature of 36.5 degrees C. After the hypoxic exposure, the animals were separated into three groups and placed for 3 h in temperature-controlled incubators set at 32 degrees C, 35 degrees C, and 38 degrees C. In Study 1, the influence of post-insult temperature modulation was assessed after graded cerebral hypoxic-ischemic injury. Brain damage was assessed 1 week after the insult by comparison of wet weights in the cerebral hemispheres ipsilateral and contralateral to the carotid artery ligation. Rectal temperatures of the animals significantly correlated with extent of brain injury after 60 min (Spearman correlation coefficient, p = 0.44, P = 0.005) and 90 min (p = 0.46, P = 0.004) but not 120 min of hypoxia (p = 0.18, P = 0.46). In Study 2, animals were exposed to 75 min hypoxia, and injury was assessed morphometrically and histologically at 1 and 4 weeks after the injury. Rectal temperatures significantly correlated with the extent of ischemic injury in the cerebral cortex (p = 0.3, P = 0.046) and striatum (p = 0.3, P = 0.048) at 1 week, but not 4 weeks, after the insult. The findings indicate that post-insult hypothermia delayed the expression of mild to moderate brain damage by more than a week, after which the damage was as severe as in normothermic animals. The results indicate that the events that determine the final expression of a neonatal hypoxic-ischemic insult can be extended over a long interval by post-insult hypothermia.

Experimental neuronal injury in the newborn lamb: a comparison of N-methyl-D-aspartic acid receptor blockade and nitric oxide synthesis inhibition on lesion size and cerebral hyperemia.

The purpose of this study was to compare the effects of dizocilipine maleate (MK-801) and NG-nitro-L-arginine methyl ester (L-NAME) on focal excitotoxic brain injury and associated hemodynamic response in the newborn lamb. A 27 gauge needle was placed into the right striatum in 28 anesthetized newborn lambs. Seven animals were placed in each group. A negative control group received 0.2 mL of buffered saline, a positive control group received 5 mumol of N-methyl-D-aspartic acid (NMDA) alone, and two groups received NMDA and pretreatment with L-NAME. Ultrasound images and cerebral blood flow determinations (microspheres) were obtained before, and at 20, 40, and 60 min after, intrastrial injection. Three animals in each group underwent histopathologic evaluation. Sonographic lesions were visible immediately after intracerebral injection. Saline injection resulted in small lesions (mean volume; 13.6 +/- 5 mm3) without hyperemia. NMDA alone resulted in larger lesions (92.9 +/- 24 mm3) and hyperemia to both hemispheres, whereas pretreatment with MK-801 reduced lesion size (11.7 +/- 6 mm3) and completely ablated cerebral hyperemia. Pretreatment with L-NAME showed no effect on lesion size (69.9 +/- 20 mm3) and hyperemia only in the ipsilateral hemisphere. Sonographic lesions correlated well with gross and histopathologic appearance. We concluded that NMDA-induced focal brain injury and associated hyperemia in the newborn lamb appear to be specific NMDA receptor-mediated events. NO production probably does not play a major part in NMDA-induced neonatal neuronal injury, and may be only partly responsible for regional hyperemia during NMDA injection.

Hypoxic and ischemic central nervous system disorders in infants and children.

Brain damage from severe hypoxemia and ischemia is involved in many childhood disorders that produce permanent disability. Recent progress in diagnosis and brain imaging as well as in the laboratory has expanded understanding of the pathophysiology of these disorders. They are currently thought to trigger a neurotoxic biochemical cascade that produces permanent cell death over a period of hours to days. A prominent feature of this cascade is synaptic dysfunction and overactivation of excitatory amino acid receptors that carry a majority of the excitatory messages transmitted in the brain. In premature infants the periventricular white matter is especially vulnerable, whereas neuronal structures are more vulnerable at term and at older ages. Numerous drugs are now known to protect the brain from neuronal damage in laboratory models. Application of this new pharmacology will require techniques to monitor cerebral metabolism and blood flow at the bedside in order to ensure that only high-risk infants are included. Although the new medications can be expected to have significant adverse effects as well as benefits, it seems likely that this therapy will be applied to certain high-risk groups over the next decade.

Quinolinate-induced injury is enhanced in developing rat brain.

Quinolinate, a metabolite of tryptophan in the kynurenine pathway, has been hypothesized to play a role in neuronal injury through activation of the N-methyl-D-aspartate (NMDA) receptor. We evaluated the ontogeny and neuroprotective pharmacology of quinolinate-induced injury in the immature rat brain. Unilateral striatal microinjections of quinolinate (150 nmol/0.5 microliter) were performed at seven ages between postnatal day (PND) 1 and 90. Injury was assessed by comparing the cross-sectional areas of the cerebral hemispheres ipsilateral and contralateral to the injection site in Nissl-stained coronal sections. The susceptibility to quinolinate-induced injury was enhanced in the immature brain with peak toxicity at PND 7 when the ipsilateral cerebral hemisphere was reduced by 16.1 +/- 3.2%. In a dose-response comparison with NMDA-induced injury at PND 7, quinolinate injury was directly related to the dose injected (r2 = 0.73, P < 0.0001), but the neurotoxicity of quinolinate was 20-times less potent than NMDA. In the PND 7 rat brain, quinolinate-induced injury was completely blocked by MK-801 (1 mg/kg, i.p.) and CGS-19755 (10 mg/kg). Dextromethorphan (20 mg/kg) and dextrorphan (20 mg/kg) were partially protective. Ifenprodil, carbamazepine, and nifedipine did not significantly protect against quinolinate-induced injury. Finally, pretreatment with MK-801 (1 mg/kg) 24 h before intracerebral injection of quinolinate resulted in greater injury compared to controls. The findings indicate that quinolinate-induced injury is enhanced in the immature brain in a pattern that is similar to NMDA-induced injury.

Ischemic stroke syndromes in childhood.

Cerebral ischemic injury is uncommon in children, but the effects are long-lasting with significant implications for the child's development. The precipitating event in ischemic infarct is generally occlusion of the cerebral vessels. This occlusion may the result of direct injury of the cerebral vasculature, thrombus formation, or emboli from more distant sources. A wide variety of conditions are known to predispose to cerebral infarcts in children. However, even in recent studies, the underlying condition is unknown in as many as half the children who suffer an ischemic stroke. To care for these children effectively, it is imperative that extensive evaluations be performed to determine the cause of the cerebral infarct. Furthermore, increasing attention will need to be directed toward the metabolic events of cerebral ischemia. A better understanding of these mechanisms may provide clues to some of the causes of ischemic injury and should lead to more effective treatments.

Susceptibility of brain to AMPA induced excitotoxicity transiently peaks during early postnatal development.

The excitatory and excitotoxic actions of the endogenous excitatory amino acid (EAA) neurotransmitter, glutamate, are mediated by activation of three common subtypes of EAA receptors: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/quisqualate and kainate receptors. EAA neurotransmitter systems play a number of physiological roles in the regulation and organization of neural systems during development. However, excessive activation of this neurotransmitter system is also implicated in the pathophysiology of several forms of acute and chronic brain injury. In this study, the susceptibility of the developing rat brain to AMPA/quisqualate receptor mediated injury was examined at eight postnatal ages (1-90 days). The receptor agonists, AMPA (25 nmol) or quisqualate (100 nmol), were stereotaxically microinjected unilaterally into the anterior striatum. The severity of resulting brain injury was assessed 5 days later by comparison of reductions in regional cortical and striatal cross-sectional areas. Microinjection of AMPA (25 nmol) produced widespread unilateral forebrain injury in the intermediate postnatal period (days 5-28). The severity of injury resulting from microinjection of a fixed dose of AMPA (25 nmol) transiently exceeded the severity of injury in adults between PND 5-28 with peak sensitivity occurring near PND 10. At PND 1, microinjection of AMPA produced a 24.5 +/- 1.7% reduction in striatal cross-sectional area, which is similar to the response observed in adult animals, and the lesion was confined to the injection site. Susceptibility to AMPA toxicity increased 2-fold from PND 1 to PND 5. At PND 10, the age of maximal sensitivity, the excitotoxic reaction to AMPA extended throughout the entire cerebral hemisphere and the mean striatal cross-sectional area was reduced by 81.7 +/- 3.9%. With advancing postnatal age, the severity of injury progressively diminished and the lesion became confined to the injection site. The developmental pattern of sensitivity to AMPA toxicity in other brain regions differed although peak sensitivity consistently occurred near PND 10. Microinjection of quisqualate produced a developmental pattern of striatal susceptibility similar to AMPA although quisqualate was a considerable less potent neurotoxin. In additional experiments, the in vivo pharmacology of AMPA and quisqualate mediated brain injury was evaluated in a PND 7 rat model in order to determine the neurotoxic characteristics and specificity of these agonists in vivo. The severity of brain injury was assessed 5 days after intrastriatal excitotoxin injection by comparison of cerebral hemisphere weights.(ABSTRACT TRUNCATED AT 400 WORDS)

The severity of excitotoxic brain injury is dependent on brain temperature in immature rat.

The effect of brain temperature on the severity of excitotoxic brain injury was evaluated in perinatal rats. Postnatal day (PND) 7 rats received unilateral intrastriatal injections of 25 nmol N-methyl-D-aspartate (NMDA) and then were exposed for 2 h to one of six different ambient temperatures. Animals were sacrificed 5 days later and the severity of brain injury was assessed quantitatively by comparison of the weights of the injected and contralateral cerebral hemispheres. Injection of NMDA consistently produced extensive unilateral brain injury in rats maintained at normothermia (36 degrees C ambient temperature; 29 +/- 1.8% reduction in the weight of the injected hemisphere). In the range of ambient temperatures between 25 degrees C and 40 degrees C, there was a linear relationship between temperature and the severity of NMDA-induced injury (r2 = 0.95, P less than 0.001, linear regression). In a separate analysis, in PND 7 rats, a positive linear relationship between ambient temperature (28 degrees C and 40 degrees C) and brain temperature was observed (r2 = 0.96, P less than 0.001). These data suggest that the severity of excitotoxic brain injury is dependent upon brain temperature.

The selective ionotropic-type quisqualate receptor agonist AMPA is a potent neurotoxin in immature rat brain.

Direct unilateral intrastriatal stereotaxic injections of the selective ionotropic-type quisqualate receptor agonist, AMPA, in postnatal day 7 rats produced tonic-clonic seizure activity. Quantitative analysis of the severity of brain injury was assessed by comparison of the disparities in the weights of injected and contralateral cerebral hemispheres 3 days after the excitotoxin injection. The amount of AMPA that produced half-maximal brain injury was 9.5 nmol as assessed by comparison of disparities in cerebral hemisphere weights. In contrast, quisqualate was 26 times less potent. The marked susceptibility of the developing rat brain to AMPA toxicity may provide a useful model to assess the neuroprotective effectiveness and selectivity of ionotropic quisqualate receptor antagonists.

The influence of growth retardation on perinatal hypoxic-ischemic brain damage.

The effect of growth retardation on the extent of brain damage produced by hypoxia-ischemia was assessed in immature rats. Newborn rats were raised in litters of 6 or 14 pups from day 2 to 7. On postnatal day 7, those immature rats raised in litters of 14 weighed 18% less than animals raised in litters of 6 (P less than 0.001). They then were subjected to cerebral hypoxia-ischemia by unilateral common carotid artery ligation followed by 3 h of exposure to 8% oxygen-92% nitrogen at 37 degrees C. Upon return to their dams, all litters were culled to 6 pups. At 30 days of age, the animals underwent perfusion-fixation of their brains under pentobarbital anesthesia. Brain damage was assessed by measuring the length and width of each cerebral hemisphere. The extent of brain damage varied from no difference in the size of the two cerebral hemispheres to marked shrinkage of the hemisphere ipsilateral to the common carotid artery occlusion. The range of brain damage between the well-nourished and poorly nourished animals was comparable. Rank order of the extent of damage demonstrated significantly greater tissue injury in those animals well nourished prior to hypoxia-ischemia (Mann-Whitney U-test; P = 0.003). The results indicate that nutritional deprivation in the immature rat is associated with a decreased rather than increased susceptibility to brain damage arising from hypoxia-ischemia. The findings of the investigation have relevance to the human infant suffering from intrauterine growth retardation (IUGR).