Faye S. Silverstein

Neurotoxicity of N-methyl-d-aspartate is markedly enhanced in developing rat central nervous system

The neurotoxic lesion produced by direct injection of 25 nmol ofN-methyl-d-aspartate (NMDA) into the corpus striatum of 7-day-old rats was compared to the effects of injecting 75 nmol into the striatum or hippocampus of adults. The area of histopathology in the immature striatum was 21 × larger than the striatal lesions in adults. Damage from NMDA injected into the immature striatum also extended into the dorsal hippocampus and produced an area of destruction which was 16 × larger than observed after direct injection into the adult hippocampus. Several studies have implicated excessiveN-methyl-d-aspartate receptor activation in the pathogenesis of hypopoxic-ischemic and hypoglycemic injury and our results suggest that this neurotoxic mechanism is extremely active in the immature brain.

Cerebral Hypoxia-Ischemia Stimulates Cytokine Gene Expression in Perinatal Rats

BACKGROUND AND PURPOSE We tested the hypothesis that cerebral hypoxia-ischemia selectively stimulates interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) gene expression in brain regions susceptible to irreversible injury in perinatal rats. METHODS To elicit focal hypoxic-ischemic brain injury, 7-day-old perinatal (P7) rats were subjected to right carotid artery ligation followed by 3 hours of 8% O2 exposure and were killed 0 to 48 hours after hypoxia. Regional tissue IL-1 beta and TNF-alpha mRNA content were measured by reverse transcription followed by polymerase chain reaction amplification (RT-PCR) in samples prepared from cortex and hippocampus of the lesioned and contralateral hemispheres. cDNAs were amplified with primers specific for IL-1 beta, TNF-alpha, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which served as an internal control. The RT-PCR products were subjected to Southern blot analysis and hybridized with 32P-labeled gene-specific probes. Radioactivity was measured in excised bands, and results were normalized on the basis of levels of GAPDH expression. RESULTS In unlesioned P7 brain, IL-1 beta mRNA was barely detectable. In lesioned forebrain, there was a marked, transient stimulation of IL-1 beta mRNA expression, peaking at 4 hours after hypoxia. Hybridization signal was increased 16- to 30-fold over values from contralateral hemisphere samples in three independent assays (P < .05 comparing values in left and right cortex and in left and right hippocampus with the Kruskal-Wallis ranking test); by 24 hours after hypoxia, levels returned to normal. Similar transient increases in TNF-alpha mRNA expression were detected. In a closely related model of perinatal brain injury elicited by focal intracerebral N-methyl-D-aspartate injection, there was a corresponding acute stimulation of IL-1 beta and TNF-alpha mRNA expression at 4 hours after injection. CONCLUSIONS These results suggest that IL-1 beta and TNF-alpha may play important roles in the response of the developing brain to acute hypoxic-ischemic injury.

MK-801 protects the neonatal brain from hypoxic-ischemic damage

Enhanced synaptic release of excitatory amino acid neurotransmitters may contribute to brain injury from hypoxia-ischemia (Meldrum, 1985; Simon et al., 1984). To examine this hypothesis in neonatal brain we tested MK-80I , a novel noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, using an in vivo experimental model of hypoxic-ischemic forebrain injury (Johnston, 1983). To induce injury, seven day old rats were anesthetized with ether and the right carotid was ligated (N = 63), after which they recovered with the dam for 2 h. The pups were then placed in a warmed (37°C) chamber and exposed to 8% oxygen balance nitrogen for 3 h, a procedure which resulted in hemispheric necrosis ipsilateral to the side of ligation. Effect of treatment was assessed using three MK-801 treatment protocols: administration of 1 dose of MK-801 (1 m g / k g ) before hypoxia (N = 10), a single dose during hypoxia (N = 18), or two doses, one before and another after 1.25 h of hypoxia (N = 9). In the second group, the timing of treatment was varied to determine if there was a critical time threshold for efficacy: a single dose was given after 1.25 h (N = 5), 1.5 h (N = 6) or 2.5 h (N = 7). Pups received one or two intraperitoneal injections of M K = 801 (N = 37) or phosphate buffered saline (N = 26); a group of untreated litter-mates served as additional controls (N = 5). Pups were sacrificed 5 days after

Therapeutic Hypothermia after Out-of-Hospital Cardiac Arrest in Children

BACKGROUND Therapeutic hypothermia is recommended for comatose adults after witnessed out-of-hospital cardiac arrest, but data about this intervention in children are limited. METHODS We conducted this trial of two targeted temperature interventions at 38 childrens hospitals involving children who remained unconscious after out-of-hospital cardiac arrest. Within 6 hours after the return of circulation, comatose patients who were older than 2 days and younger than 18 years of age were randomly assigned to therapeutic hypothermia (target temperature, 33.0°C) or therapeutic normothermia (target temperature, 36.8°C). The primary efficacy outcome, survival at 12 months after cardiac arrest with a Vineland Adaptive Behavior Scales, second edition (VABS-II), score of 70 or higher (on a scale from 20 to 160, with higher scores indicating better function), was evaluated among patients with a VABS-II score of at least 70 before cardiac arrest. RESULTS A total of 295 patients underwent randomization. Among the 260 patients with data that could be evaluated and who had a VABS-II score of at least 70 before cardiac arrest, there was no significant difference in the primary outcome between the hypothermia group and the normothermia group (20% vs. 12%; relative likelihood, 1.54; 95% confidence interval [CI], 0.86 to 2.76; P=0.14). Among all the patients with data that could be evaluated, the change in the VABS-II score from baseline to 12 months was not significantly different (P=0.13) and 1-year survival was similar (38% in the hypothermia group vs. 29% in the normothermia group; relative likelihood, 1.29; 95% CI, 0.93 to 1.79; P=0.13). The groups had similar incidences of infection and serious arrhythmias, as well as similar use of blood products and 28-day mortality. CONCLUSIONS In comatose children who survived out-of-hospital cardiac arrest, therapeutic hypothermia, as compared with therapeutic normothermia, did not confer a significant benefit in survival with a good functional outcome at 1 year. (Funded by the National Heart, Lung, and Blood Institute and others; THAPCA-OH ClinicalTrials.gov number, NCT00878644.).

Neonatal hypoxic-ischemic injury increases forebrain subventricular zone neurogenesis in the mouse.

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ)-olfactory bulb pathway and increases in the adult after brain insults. The influence of neonatal injury on SVZ neural precursors is unknown. We examined the effects of hypoxia-ischemia (HI) on neonatal mouse SVZ cell proliferation and neurogenesis. Postnatal day 10 (P10) mice underwent right carotid artery ligation followed by 10% O2 exposure for 45 min. The SVZ area and hemispheric injury were quantified morphometrically 1-3 weeks later. Bromodeoxyuridine (BrdU) was used to label proliferating cells, and cell phenotypes of the progeny were identified by immunohistochemistry. HI significantly enlarged the ipsilateral SVZ at P18, P24, and P31, and increases in the SVZ area correlated directly with the degree of hemispheric damage. HI also stimulated cell proliferation and neurogenesis in the SVZ and peri-infarct striatum. Some newborn cells expressed a neuronal phenotype at P24, but not at P31, indicating that neurogenesis was short-lived. These results suggest that augmenting SVZ neuroblast recruitment and survival may improve neural repair after neonatal brain injury.

CYTOKINES AND PERINATAL BRAIN INJURY

A rapidly expanding body of data provides support for the hypothesis that pro-inflammatory cytokines including interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) are expressed acutely in injured brain and contribute to progressive neuronal damage. Little is known about the pathogenetic role of these cytokines in perinatal brain injury. Recent experimental studies have incorporated two closely related in vivo perinatal rodent brain injury models to evaluate the role(s) of pro-inflammatory cytokines in the progression of neuronal injury: a perinatal stroke model, elicited by unilateral carotid artery ligation and subsequent timed exposure to 8% oxygen in 7-day-old rats, and a model of excitotoxic injury, elicited by stereotactic intra-cerebral injection of the selective excitatory amino acid agonist NMDA. Each of these lesioning methods results in reproducible, quantifiable focal forebrain injury at this developmental stage. Acute brain injury, evoked by cerebral hypoxia-ischemia or excitotoxin lesioning, results in transient marked increases in expression of IL-1 beta, and TNF-alpha mRNA in brain regions susceptible to irreversible injury, and there is evidence that pharmacological antagonism of IL-1 receptors can attenuate injury in both models. Recent studies also suggest that complementary strategies, based on pharmacological antagonism of platelet activating factor and on neutrophil depletion can also limit the extent of irreversible injury. In summary, current data suggest that pro-inflammatory cytokines contribute to the progression of perinatal brain injury, and that these mediators are important targets for neuroprotective interventions in the acute post-injury period.

Mice Deficient in Interleukin-1 Converting Enzyme Are Resistant to Neonatal Hypoxic-Ischemic Brain Damage

Interleukin-1 (IL-1) converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1β to active IL-1β. The pro-inflammatory cytokine IL-1β is implicated as a mediator of hypoxic-ischemic (HI) brain injury, both in experimental models and in humans. ICE is a member of a family of ICE-like proteases (caspases) that mediate apoptotic cell death in diverse tissues. The authors hypothesized that in neonatal mice with a homozygous deletion of ICE (ICE-KO) the severity of brain injury elicited by a focal cerebral HI insult would be reduced, relativefto wild-type mice. Paired litters of 9- to 10-day-old ICE-KO and wild-type mice underwent right carotid ligation, followed by 70 or 120 minutes of exposure to 10% O2, In this neonatal model of transient focal cerebral ischemia followed by reperfusion, the duration of hypoxia exposure determines the duration of cerebral ischemia and the severity of tissue damage. Outcome was evaluated 5 or 21 days after lesioning; severity of injury was quantified by morphometric estimation of bilateral cortical; striatal, and dorsal hippocampal volumes. In animals that underwent the moderate HI insult (70-minute hypoxia), damage was attenuated in ICE-KO mice, when evaluated at 5 or 21 days post-lesioning. In contrast, in mice that underwent the more severe HI insult (120-minute hypoxia), injury severity was the same in both groups. Reductions in intra-HI CBF, measured by laser Doppler flowmetry, and intra- and post-HI temperatures did not differ between groups. These results show that ICE activity contributes to the progression of neonatal HI brain injury in this model. Whether these deleterious effects are mediated by proinflammatory actions of IL-lβ and/or by pro-apoptotic mechanisms is an important question for future studies.

Magnesium reduces N-methyl-d-aspartate (NMDA)-mediated brain injury in perinatal rats

We evaluated the neuroprotective effects of systemically administered magnesium against N-methyl-D-aspartate (NMDA)-mediated brain injury in perinatal rats. Postnatal day (PND) 7 rats received unilateral intrastriatal injections of 25 nmol NMDA followed 15 min later by single or multiple doses of magnesium intraperitoneally (i.p.). Animals were sacrificed five days later and the severity of brain injury was assessed by comparison of the weights of the injected and contralateral cerebral hemispheres. NMDA injection reduced the weight of the injected cerebral hemisphere by 31 +/- 3%. Single doses of magnesium reduced the severity of NMDA-induced brain injury in a dose-dependent fashion (2 mmol/kg, 29 +/- 11% protection; 3 mmol/kg, 52 +/- 12% protection; 4 mmol/kg, 62 +/- 7% protection). Multiple doses of magnesium reduced brain injury by 65 +/- 4%. These data demonstrate that systemically administered magnesium antagonizes the neurotoxic effects of NMDA in vivo in perinatal rats.

Hypoxic-ischemic brain injury induces an acute microglial reaction in perinatal rats.

Activated microglia may contribute to the progression of neuronal injury after a wide range of CNS insults. In this study, we used two complementary methods to evaluate acute changes in the morphology and regional distribution of microglia induced by a focal hypoxic-ischemic insult in 7-d-old (P7) rats. To elicit injury, P7 rats underwent right carotid ligation followed by 3 h of 8% O2 exposure; rats were killed 10 min to 5 d later (n ≥ 3/group). A histochemical assay using Griffonia simplicifolia B4-isolectin enabled detection of both resting and activated microglia in tissue sections; vascular cells were also reactive. Activated microglia were also identified immunocytochemically using a macrophage-specific MAb, ED-1. In normal P7-12 brain, lectin, and ED-1 immunoreactive-activated microglia were concentrated in white matter; lectin-positive resting, ramified microglia were also detected throughout the gray and white matter. Subtle morphologic evidence of microglial activation was noted 10 min posthypoxia-ischemia in the lesioned right cerebral hemisphere; activated microglia began to accumulate within the next 4 h. Accumulation of lectin-positive activated microglia peaked at 2-4 d posthypoxia-ischemia. ED-1 immunoreactive-microglia were first noted 4 h after hypoxic-ischemic injury in the lesioned right hemisphere, and there was a corresponding increase in accumulation over the first 48 h posthypoxia-ischemia. In the left hemisphere, contralateral to the ligation, no increase in activated microglia were detected with either method. In brain sections where no neuronal injury was evident, activated microglia did not accumulate. These data demonstrate that perinatal hypoxicischemic brain injury induced rapid accumulation of activated microglia in hypoxic-ischemic forebrain.There is increasing evidence that phagocytic cells, including microglia and macrophages, respond acutely to diverse forms of CNS injury. In the adult CNS, ischemic and excitotoxic injury stimulate a well characterized microglial response that includes both changes in morphology and increased accumulation in regions of injury(1–9). Considerable data indicate that, in addition to phagocytosis of degenerating elements, these cells secrete a wide range of soluble factors, such as cytokines (10–13), substances with excitatory amino acid agonist properties (14), and glial promoting factors (10, 11), that may influence the extent of neuronal injury, axonal growth, synaptic plasticity, and astroglial hyperplasia. Microglia also play important roles in normal CNS development(15), and activated microglia are detectable in normal immature rodent brain; yet, little is known about the response of these cells after injury in the developing brain. In the only study that has attempted to compare the microglial response in the developing and adult brain(1), lesions in the adult visual cortex induced slower cell death and a much more protracted phagocytic response than in younger animals.

Topiramate Extends the Therapeutic Window for Hypothermia-Mediated Neuroprotection After Stroke in Neonatal Rats

Background and Purpose— Critical factors influencing the neuroprotective efficacy of postischemic hypothermia include depth, duration, and time of onset of cooling. In clinical practice, there is an unavoidable lag between the hypoxic-ischemic (HI) insult and the opportunity to initiate cooling. We hypothesized that early administration of a neuroprotective agent in combination with later-onset cooling could represent an effective therapeutic intervention after neonatal HI. We evaluated whether treatment with topiramate, a clinically available anticonvulsant, increased the efficacy of delayed post-HI hypothermia in a neonatal rat stroke model. Methods— Postnatal day 7 (P7) rats underwent right carotid artery ligation followed by 1.5 hours of exposure to 8% oxygen. Fifteen minutes post-HI, animals received injections of topiramate (30 mg/kg) or PBS. Cooling was initiated 3 hours later (“delayed hypothermia”) in all animals (3 hours, in 27°C incubator). Functional outcome (forepaw response to vibrissae stimulation) and pathology (morphometric lesion measurements) were evaluated at P15 and P35. Results— Neither topiramate nor delayed hypothermia alone conferred protection in this protocol. Combined treatment with topiramate and delayed hypothermia improved both performance and pathological outcome in P15 and P35 rats compared with PBS-treated animals that underwent delayed hypothermia concurrently. At P15, functional measures were better in topiramate-treated animals (mean correct forepaw response 9.3/10 versus 4.8/10; P< 0.001), and there was >50% reduction in tissue loss (P< 0.001); trends were similar at P35. Conclusions— Our data provide the impetus for further evaluation of therapeutic approaches that combine drug therapy with delayed-onset cooling after neonatal HI brain injury.