Attenuation of hypoxia-ischemia-induced monocyte chemoattractant protein-1 expression in brain of neonatal mice deficient in interleukin-1 converting enzyme.

Interleukin-1beta (IL-1beta) upregulates expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in many experimental models. In neonatal rodent brain, hypoxia-ischemia rapidly stimulates expression of this chemokine, although the role of IL-1beta in regulating this response is unknown. Interleukin-1 converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1beta to generate mature IL-1beta. Neonatal mice with a homozygous deletion of ICE (ICE -/-) are resistant to moderate, but not to severe cerebral hypoxic-ischemic insults, relative to their wild-type controls. We hypothesized that their resistance to moderate hypoxic-ischemic insults is mediated by suppression of the acute inflammatory response to brain injury in the absence of IL-1beta, and that hypoxia-ischemia induced MCP-1 expression would be attenuated in ICE -/- animals. To test this hypothesis, paired litters of 9-10-day-old ICE -/- and wild-type mice underwent right carotid ligation, followed by 40, 70 or 120 min exposure to 10% O2 and ischemia-induced changes in MCP-1 mRNA and protein were compared, using a semi-quantitative reverse-transcription polymerase chain reaction assay and an ELISA, respectively. With a lesioning protocol that elicits minimal injury in wild-types (ligation+40 min 10% O2), there was an attenuation of hypoxia-ischemia-induced MCP-1 production at 8 h post-hypoxia; in contrast, in animals that underwent longer periods of hypoxia-ischemia the magnitude of injury-induced induced MCP-1 production did not differ between wild-type and ICE -/- animals. These results demonstrate both that the acute inflammatory response to hypoxia-ischemia is attenuated in ICE -/- animals, and also that hypoxic-ischemic brain injury stimulates MCP-1 expression even in the absence of IL-1beta activity.

Pentoxifylline attenuates hypoxic-ischemic brain injury in immature rats.

Inflammatory mediators are implicated in the pathogenesis of ischemic injury in immature brain. The phosphodiesterase inhibitor pentoxifylline inhibits production of tumor necrosis factor-alpha and platelet-activating factor. We hypothesized that pentoxifylline treatment would attenuate hypoxic-ischemic brain injury in immature rats. Seven-day-old rats (n = 79) underwent right carotid ligation, followed by hypoxia (FiO2 = 0.08). Rats received pentoxifylline immediately before and again after hypoxia (two doses, 25-150 mg/kg/dose, n = 34), or vehicle (n = 27). In separate experiments, rats received pentoxifylline treatment (40 mg/kg/dose, n = 8), or vehicle (n = 10) immediately and again 3 h after hypoxia-ischemia. Severity of injury was assessed 5 d later by visual evaluation of ipsilateral hemisphere infarction and by measurement of bilateral hemispheric cross-sectional areas. Pentoxifylline pretreatment reduced the incidence of liquefactive cerebral infarction, from 75% in controls to 10% with pentoxifylline, 40 mg/kg/dose (p<0.001, chi2 trend test). Quantification of hemispheric areas confirmed these findings. In contrast, posthypoxic-ischemic treatment with pentoxifylline resulted in only a modest reduction in cortical damage, without an overall reduction in incidence of infarction. Phosphodiesterase inhibition may be an effective strategy to use to decrease the severity of neonatal hypoxic-ischemic brain injury. Pretreatment regimens could be clinically relevant in settings in which an increased risk of cerebral ischemia can be anticipated, such as in infants undergoing surgery to correct congenital heart disease.

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-1beta to active IL-1beta. The pro-inflammatory cytokine IL-1beta 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, relative to 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 flow-metry, 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 pro-inflammatory actions of IL-1beta and/or by pro-apoptotic mechanisms is an important question for future studies.

Tumor necrosis factor-a attenuates N-methyl-D-aspartate-mediated neurotoxicity in neonatal rat hippocampus.

Tumor necrosis factor-a TNFa. has been implicated in the pathophysiology of acute neonatal brain injury. We hypothesized that acute brain injury would induce TNFa expression and that exogenous TNFa would influence the severity of N-methyl-D-aspartate-induced tissue damage. We performed two complementary groups of experiments to evaluate the potential role s. of TNFa in a neonatal rodent model of excitotoxic injury, elicited by intracerebral injection of N-methyl-D-aspartate. We used immunohistochemistry and ELISA to evaluate N-methyl-D-aspartate-induced changes in TNFa expression, and we co-injected TNFa with N-methyl-D-aspartate, to evaluate the effect of this cytokine on the severity of tissue injury. Both intra-hippocampal and intra-striatal injection of N-methyl-D-aspartate 5 nmol. stimulated TNFa expression. Increased TNFa expression was detected 3-12 h after lesioning; TNFa was localized both in glial cells in the corpus callosum, and in cells with the morphology of interneurons in the ipsilateral hippocampus, striatum, cortex and thalamus. Intra-hippocampal or intra-striatal administration of TNFa 50 ng. alone did not elicit neuropathologic damage. In the hippocampus, when co-injected with N-methyl-D-aspartate 5 or 10 nmol., TNFa 50 ng. attenuated excitotoxic injury by 35%-57%, compared to controls co-injected with heat-treated TNFa. In contrast, in the striatum, co-injection of TNFa with N-methyl-D-aspartate had no effect on the severity of the ensuing damage. The data indicate that TNFa is rapidly produced in glial cells and neurons after an excitotoxic insult in the neonatal rat brain, and that administration of exogenous TNFa results in region-specific attenuation of excitotoxic damage. We speculate that endogenous TNFa may modulate the tissue response to excitotoxic injury in the developing brain.

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.

Adenovirus-mediated over-expression of interleukin-1 receptor antagonist reduces susceptibility to excitotoxic brain injury in perinatal rats.

In seven-day-old rats, intracerebral injection of N-methyl-D-aspartate transiently stimulates expression of Interleukin-1 beta messenger RNA. To evaluate the role of Interleukin-1 beta in the pathogenesis of excitotoxic injury, we sought to determine if Interleukin-1 receptor antagonist, an endogenous competitive inhibitor of Interleukin-1 beta, could attenuate N-methyl-D-aspartate-induced injury. To induce sustained over-expression of Interleukin-1 receptor antagonist in the brain, a recombinant adenovirus encoding Interleukin-1 receptor antagonist was administered by intracerebroventricular injection into three-day-old rats. Increased brain concentrations of Interleukin-1 receptor antagonist two to six days later were documented by assays of tissue homogenates and by immunocytochemistry. To evaluate the impact of Interleukin-1 receptor antagonist on N-methyl-D-aspartate neurotoxicity, three-day-old animals received intracerebroventricular injections of either adenovirus encoding Interleukin-1 receptor antagonist or a control adenovirus encoding beta-galactosidase, followed four days later by right intrastriatal injections of N-methyl-D-aspartate (10 nmol/0.5 microliter), a dose that typically elicits excitotoxic injury in the ipsilateral striatum and adjacent hippocampus, or saline. Animals were killed five days later, and brain damage was quantitated by measurement of bilateral cross-sectional areas of the striatum and anterior hippocampus. In three independent experiments, in N-methyl-D-aspartate-lesioned animals, both striatal and hippocampal injuries were reduced in animals that had been infected with adenovirus that encoded Interleukin-1 receptor antagonist, in comparison with littermates infected with the control adenovirus (right striatal volume loss ranged from 16 to 24%, compared with 54-65% volume loss in control). There was no striatal atrophy in adenovirus-infected saline-injected animals. These results provide strong support for the hypothesis that Interleukin-1 beta is a mediator of excitotoxic brain injury in perinatal rats.

Potentiation of N-methyl-D-aspartate-mediated brain injury by a human immunodeficiency virus-1-derived peptide in perinatal rodents.

In this study, we tested the hypothesis that human immunodeficiency virus (HIV)-1-derived peptides augment the neurotoxicity of excitatory amino acid agonists in vivo in postnatal day (PND) 7 rats. Stereotaxic intracerebral injections of the excitatory amino acid agonist N-methyl-D-aspartate (NMDA), alone or coinjected with an HIV-derived recombinant fusion peptide envelope gag (env-gag) were performed in PND-7 rats [group I: 5 nmol NMDA, n = 20; group II: 5 nmol NMDA + low-dose (1 or 50 ng) env-gag, n = 27; group III: 5 nmol NMDA + high-dose (100 ng) env-gag, n = 20], and brain injury was evaluated on PND 12. Based on histopathology scoring and measurements of hippocampal cross-sectional areas in the injected and contralateral hemispheres, coinjection of 100 ng of env-gag with 5 nmol of NMDA markedly increased the severity of resulting injury (p < 0.002, comparing histopathology scores; p < 0.003, comparing interhemispheric differences in hippocampal areas). These data suggest that in the developing nervous system HIV neurotoxicity may result, at least in part, from overactivation of excitatory amino acid receptors, and that perinatal rodent models may provide clinically relevant insights about the pathophysiology of HIV-mediated brain injury.