Tumor necrosis factor-α attenuates N-methyl-d-aspartate-mediated neurotoxicity in neonatal rat hippocampus

Tumor necrosis factor-α (TNFα) has been implicated in the pathophysiology of acute neonatal brain injury. We hypothesized that acute brain injury would induce TNFα expression and that exogenous TNFα 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 TNFα 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 TNFα expression, and we co-injected TNFα 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 TNFα expression. Increased TNFα expression was detected 3–12 h after lesioning; TNFα 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 TNFα (50 ng) alone did not elicit neuropathologic damage. In the hippocampus, when co-injected with N-methyl-d-aspartate (5 or 10 nmol), TNFα (50 ng) attenuated excitotoxic injury by 35%–57%, compared to controls co-injected with heat-treated TNFα. In contrast, in the striatum, co-injection of TNFα with N-methyl-d-aspartate had no effect on the severity of the ensuing damage. The data indicate that TNFα is rapidly produced in glial cells and neurons after an excitotoxic insult in the neonatal rat brain, and that administration of exogenous TNFα results in region-specific attenuation of excitotoxic damage. We speculate that endogenous TNFα may modulate the tissue response to excitotoxic injury in the developing brain.     

Acute excitotoxic injury induces expression of monocyte chemoattractant protein-1 and its receptor, CCR2, in neonatal rat brain

Chemokines are a family of structurally related cytokines that activate and recruit leukocytes into areas of inflammation. The "CC" chemokine, monocyte chemoattractant protein (MCP)-1 may regulate the microglia/monocyte response to acute brain injury. Recent studies have documented increased expression of MCP-1 in diverse acute and chronic experimental brain injury models; in contrast, there is little information regarding expression of the MCP-1 receptor, CCR2, in the brain. In the neonatal rat brain, acute excitotoxic injury elicits a rapid and intense microglial response. To determine if MCP-1 could be a regulator of this response, we evaluated the impact of excitotoxic injury on MCP-1 and CCR2 expression in the neonatal rat brain. We used a reproducible model of focal excitotoxic brain injury elicited by intrahippocampal injection of NMDA (10 nmol) in 7-day-old rats, to examine injury-induced alterations in MCP-1 and CCR2 expression. RT-PCR assays demonstrated rapid stimulation of both MCP-1 and CCR2 mRNA expression. MCP-1 protein content, measured by ELISA in tissue extracts, increased >30-fold in lesioned tissue 8-12 h after lesioning. CCR2 protein was also detectable in tissue extracts. Double-immunofluorescent labeling enabled localization of CCR2 both to activated microglia/monocytes in the corpus callosum adjacent to the lesioned hippocampus and subsequently in microglia/monocytes infiltrating the pyramidal cell layer of the lesioned hippocampus. These results demonstrate that in the neonatal brain, acute excitotoxic injury stimulates expression of both MCP-1 and its receptor, CCR2, and suggests that MCP-1 regulates the microglial/monocyte response to acute brain injury.     

Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage

Stem cell therapy is a hope for the treatment of some childhood neurological disorders. We examined whether human neural stem cells (hNSCs) replace lost cells in a newborn mouse model of brain damage. Excitotoxic lesions were made in neonatal mouse forebrain with the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA). QA induced apoptosis in neocortex, hippocampus, striatum, white matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Cells immunopositive for inducible nitric oxide synthase were present in damaged white matter and subventricular zone. Three days after injury, mice received brain parenchymal or intraventricular injections of hNSCs derived from embryonic germ (EG) cells. Human cells were prelabeled in vitro with DiD for in vivo tracking. The locations of hNSCs within the mouse brain were determined through DiD fluorescence and immunodetection of human-specific nestin and nuclear antigen 7 days after transplantation. hNSCs survived transplantation into the lesioned mouse brain, as evidenced by human cell markers and DiD fluorescence. The cells migrated away from the injection site and were found at sites of injury within the striatum, hippocampus, thalamus, and white matter tracts and at remote locations in the brain. Subsets of grafted cells expressed neuronal and glial cell markers. hNSCs restored partially the complement of striatal neurons in brain-damaged mice. We conclude that human EG cell-derived NSCs can engraft successfully into injured newborn brain, where they can survive and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. (c) 2005 Wiley-Liss, Inc.     

Quantitative assessment of neuroprotection against NMDA-induced brain injury

In immature rodent brain, unilateral intrastriatal injections of selected excitatory amino acid (EAA) receptor agonists, such as N-methyl-D-aspartate (NMDA), produce prominent ipsilateral forebrain lesions. In Postnatal Day (PND) 7 rats that receive a right intrastriatal injection of NMDA (25 nmol) and are sacrificed 5 days later, there is a considerable and consistent reduction in the weight of the injected cerebral hemisphere relative to that of the contralateral side (-28.5 +/- 1.9%, n = 6). In animals treated with specific NMDA receptor antagonists, the severity of NMDA-induced damage is markedly reduced. We have previously reported that the efficacy of potential neuroprotective drugs in limiting NMDA-induced lesions can be assessed quantitatively by comparison of hemisphere weights after a unilateral NMDA injection. In this study, we compared three quantitative methods to evaluate the severity of NMDA-induced brain injury and the degree of neuroprotection provided by NMDA receptor antagonists. We characterized the severity of brain injury resulting from intrastriatal injections of 1-50 nmol NMDA in PND 7 rats sacrificed on PND 12 by (i) comparison of cerebral hemisphere weights; (ii) assay of the activity of the cholinergic neuronal marker, choline acetyltransferase (ChAT) activity; and (iii) measurement of regional brain cross-sectional areas. The severity of the resulting brain injury as assessed by comparison of hemisphere weights increased linearly with the amount of NMDA injected into the striatum up to 25 nmol NMDA. The magnitude of injury was highly correlated with the degree of reduction in ChAT activity (r2 = 0.97).(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine metabolism and glutamate receptor agonists-mediated excitotoxicity in the rat brain.

Putrescine (PUT) increases have been seen in a range of models of neuropathological disturbances. The present study was designed to compare the ability of various types of glutamate receptor agonist to promote excitotoxic brain damage and to examine whether a PUT increase is a general marker of excitotoxic brain damage. To that end, we evaluated features of brain damage associated with the excitotoxicity induced by both ionotropic glutamate receptor (iGluR) and metabotropic glutamate receptor (mGluR) agonists in the conscious rat and the changes produced in the regulation of polyamine metabolism. Intracerebroventricular infusion of N-methyl-D-aspartate (NMDA; 80 nmol), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 15 nmol), kainic acid (KA; 2.3 nmol), (R,S)-3,5-dihydroxyphenylglycine (3,5-DHPG; 1.5 micromol), and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 2 micromol) produced similar seizure incidences (76-84%) in the rat. The convulsant episodes appeared sooner after iGluR (13-22 min) than after mGluR agonists (50-179 min). Histological analysis of the hippocampus 24 hr after seizures indicated several degrees of excitotoxic injury after equiconvulsive doses of the iGluR and mGluR agonists assayed. The agonists can be placed in the following order, according to the degree of damage they produce: AMPA > 3,5-DHPG approximately KA > NMDA > 1S,3R-ACPD. In the frontal cortex, moderate to low levels of damage were observed after all GluR agonists. Both iGluR- and mGluR-induced seizures produced an overshoot in the hippocampal and cortical PUT concentration, whereas spermidine and spermine levels were similar to control. Moreover, a concurrence of increased PUT levels and brain damage was observed, indicating that PUT is a general marker of excitotoxic brain damage.     

Inhibition of TNF-alpha can attenuate or exacerbate excitotoxic injury in neonatal rat brain

Tumor necrosis factor (TNF)-alpha, a multifunctional pro-inflammatory cytokine, has been implicated in the pathogenesis of acute ischemic brain injury. Recent data also suggest that TNF-alpha is a clinically relevant mediator of neonatal brain injury. We hypothesized that inhibition of TNF-alpha activity would reduce excitotoxic brain injury in neonatal rats. To test this hypothesis, we evaluated the efficacy of a TNF binding protein (bp) in attenuating NMDA-induced injury in 7 day old rats. Intrastriatal co-injection of TNFbp (3.75 microg) with NMDA (10 nmol) reduced striatal injury by 26%; in contrast, intra-hippocampal co-injection of TNFbp (3.75 microg) with NMDA (10 nmol) increased hippocampal damage by 68%. These findings indicate that TNF-alpha may have both beneficial and deleterious effects in the injured neonatal brain.     

Neonatal mice lacking functional Fas death receptors are resistant to hypoxic-ischemic brain injury

Neonatal hypoxia-ischemia (HI) upregulates Fas death receptor expression in the brain, and alterations in expression and activity of Fas signaling intermediates occur in neonatal brain injury. B6.MRL-Tnfrsf6(lpr) mice lacking functional Fas death receptors are protected from HI brain damage in cortex, striatum, and thalamus compared to wild-type mice. Expression of Fas death receptor and active caspases increase in the cortex after HI. In wild-type mice, the hippocampus is most severely injured, and the hippocampus is the only region not protected in the B6.MRL-Tnfrsf6(lpr) mice. The selective vulnerability of the hippocampus to injury correlates with (1) lower basal expression of [Fas-associated death-domain-like IL-1beta-converting enzyme]-inhibitory protein (FLIP), (2) increased degradation of spectrin to its 145 or 150 kDa breakdown product, and (3) a higher percentage of non-apoptotic cell death following neonatal HI. We conclude that Fas signaling via both extrinsic and intrinsic caspase cascades causes brain injury following neonatal HI in a region-dependent manner. Basal levels of endogenous decoy proteins may modulate the response to Fas death receptor signaling and provide a novel approach to understanding mechanisms of neonatal brain injury.     

Excitotoxic brain damage in the rat induces interleukin-1beta protein in microglia and astrocytes: correlation with the progression of cell death

Interleukin-1 beta (IL-1beta) has been proposed as a mediator of several forms of brain damage, including that induced by excitotoxins. In vitro studies suggest that glial cells are the effector cells of IL-1beta-mediated neurodegeneration. We have investigated the expression of IL-1beta protein by glial cells in vivo in response to NMDA receptor-mediated excitotoxicity in the rat parietal cortex and striatum. Expression of IL-1beta by glial cells was investigated using immunocytochemistry 30 min to 7 days after infusion of the NMDA agonist cis-2,4-methanoglutamate (MGlu; 10 nmol) into the cortex. Early expression (1-4 h) of IL-1beta by microglia was directly related to lesion development. Later expression by microglia (up to 24 h), and by astrocytes (2-7 days), was widespread compared to the area involved in excitotoxic cell death and co-localised with areas of reactive gliosis. Infusion of MGlu into the striatum induced a similar temporal pattern of IL-1beta expression by microglia and astrocytes. However, IL-1beta-expressing glial cells were localised strictly to the area of striatal cell death. Infusion of PBS or a subtoxic dose of MGlu into the cortex or striatum induced only limited neuronal death and negligible glial IL-1beta expression. These studies reveal that IL-1beta is expressed specifically by microglia during the early response to excitotoxicity in the adult rat cortex and striatum. However, the widespread and delayed IL-1beta expression by astrocytes suggests diverse roles for IL-1beta in response to excitotoxicity.     

Glial expression of the 90-kDa heat shock protein (HSP90) and the 94-kDa glucose-regulated protein (GRP94) following an excitotoxic lesion in the mouse hippocampus

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. Although several studies have been focused on the expression of HSPs, little is known about HSP90s expression in glial cells under neuropathological conditions. In this study, we evaluated the expression pattern of the glial cell-related HSP90 and GRP94 proteins, following the induction of an excitotoxic lesion in the mouse brain. Adult mice received an intracerebroventricular injection of kainic acid; the brain tissue was then analyzed immunohistochemically for HSPs and double labeling using glial markers. HSPs expression was quantified by Western blot analysis. Excitotoxic damage was found to cause pyramidal cell degeneration in the CA3 region of the hippocampus. In the injured hippocampus, reactive microglia/macrophages expressed HSP90 from 12 h until 7 days postlesion (PL), showing maximal levels at day 1. In parallel, hippocampal reactive astrocytes showed the expression of GRP94 from 12 h until 7 days PL. In general, HSPs expression was transient, peaked at 1-3 days PL and reached basal levels by day 7. For the first time, our data demonstrate the injury-induced expression of HSP90 and GRP94 in glial cells, which may contribute to the mechanism of glial cell protection and adaptation in response to damage, thereby playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP90 may provide antioxidant protective mechanisms against microglia/macrophages, whereas GRP94 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.     

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)     

Fructose-1,6-biphosphate prevents excitotoxic neuronal cell death in the neonatal mouse brain

The excitotoxic cascade may represent an important pathway leading to brain damage and cerebral palsy. Brain lesions induced in newborn mice by ibotenate (acting on N-methyl-D-aspartate receptors) and by S-bromowillardiine (acting on alpha-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid and kainate receptors) mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage. Fructose 1,6-biphosphate (FBP) is a high-energy glycolytic pathway intermediate which, in therapeutic doses, is non-toxic and neuroprotective in hypoxic-ischemic models of brain injury. Mechanisms of action include modulation of intracellular calcium through phospholipase C (PLC) activation. The goal of this study was to determine the neuroprotective effects of FBP in a mouse model of neonatal excitotoxic brain injury. Mice that received intraperitoneal FBP had a significant reduction in size of ibotenate-induced (80% reduction) or S-bromowillardiine-induced (40% reduction) cortical plate lesions when compared with control animals. Studies of fragmented DNA and cleaved caspase 3 confirmed the survival promoting effects of FBP. FBP had no detectable effect on excitotoxic white matter lesions. The effects of FBP were antagonized by co-administration of PLC, protein kinase C or mitogen-associated protein kinase inhibitors but not by protein kinase A inhibitor. A moderate, transient cooling of pups immediately after the insult extended the therapeutic window for FBP, as FBP administered 24 h after ibotenate was still significantly neuroprotective in these pups. This data extends the neuroprotective profile of FBP in neonatal brain injury and identifies gray matter lesions involving N-methyl-D-aspartate receptors as a major target for this promising drug.     

Pharmacological manipulation of the NMDA receptor differentially protects from systemic kainic acid neuropathology: evaluation through ornithine decarboxylase induction, morphology and GFAP immunohistochemistry

The excitotoxic brain damage caused by systemic administration of kainic acid requires the activation of N-methyl-D-aspartate (NMDA) receptors in order to fully express its neurotoxic potency. We have tested the relative efficacy of different manipulations of the NMDA receptor on morphological, immunohistochemical and neurochemical parameters in this experimental model. A competitive (CGP 39551) and a non-competitive (MK 801) antagonist of the NMDA receptor, granted full protection against neuronal degeneration and consequent glial proliferation in the hippocampus and olfactory cortex, two regions severely affected by systemic administration of kainic acid. In addition, CGP 39551 completely counteracted the dramatic induction of the enzyme ornithine decarboxylase which occurs shortly after kainic acid administration. Systemic administration of high amounts of MgSO4 concomitantly and after kainic acid injection, appeared to partially prevent neuronal degeneration but had no clear effects on glial reaction and ornithine decarboxylase induction. Finally administration of an antagonist of the polyamine site present in the NMDA receptor (SL 82.0715), did not appear to have any protective effect at the dose used here. The present results help to better understand the ways by which it could be possible to counteract excitotoxic brain injuries.     

Neuroprotective effects of MK-801, TCP, PCP and CPP against N-methyl-D-aspartate induced neurotoxicity in an in vivo perinatal rat model

Three non-competitive antagonists (MK-801, TCP, PCP) and one competitive antagonist (CPP) of N-methyl-D-aspartate (NMDA) receptors, were compared for their ability to antagonize neurotoxic actions of NMDA injected into the brains of 7-day-old rats. Unilateral intracerebral injection of NMDA (25 nmol/0.5 microliters) into the corpus striatum of pups consistently produced severe confluent neuronal necrosis in the striatum extending into the dorsal hippocampus and overlying neocortex. The distribution of damage corresponded to the topography of NMDA type glutamate receptors in the vulnerable regions. With this lesion in developing brain, the weight of the injected hemisphere 5 days later can be used as a quantitative measure of brain injury. Intraperitoneal administration of MK-801 (0.02-42.0 mumol/kg), TCP (3.5-54.0 mumol/kg), PCP (1.0-41.0 mumol/kg), and CPP (1.0-60.0 mumol/kg) 15 min after NMDA injection had prominent dose-dependent neuroprotective effects. MK-801 was 14 times more potent than other compounds tested and the 50% protective dose (PD50, that dose which reduced damage by 50% relative to untreated NMDA-injected controls) was 0.63 mumol/kg. Corresponding values for CPP, PCP, and TCP were 8.84, 10.85, and 24.05 mumol/kg respectively. The lowest dose of MK-801 that provided significant protection was 0.2 mumol/kg (0.04 mg/kg, 37.9 +/- 4.6% protection). Four mumol/kg (0.8 mg/kg) of MK-801 completely protected against NMDA-mediated damage. The study provides the first direct in vivo comparison of the neuroprotective abilities of these compounds. Systemic administrations of MK-801, TCP, PCP, and CPP all limit NMDA-induced neuronal injury in this model. The susceptibility of the immature brain to the neurotoxicity of NMDA provides a sensitive, reproducible, and quantitative in vivo system for comparing the effectiveness of drugs with protective actions against excitotoxic neuronal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 3-nitropropionic acid on kynurenine aminotransferase in the rat brain

Activation of excitatory amino acid receptors by endogenous excitotoxins results in degenerative changes characteristic of neurodegenerative brain diseases such as Huntington's disease. Excitatory amino acid receptors are present in the highest concentration in the striatum, the hippocampal region, and the temporal lobe. The most potent, naturally occurring excitatory amino acid receptor antagonist is kynurenic acid (KYNA) which acts preferentially on N-methyl-D-aspartate (NMDA) receptors. KYNA is produced from L-kynurenine, by the action of the enzymes kynurenine aminotransferases (KAT I and KAT II). Several inhibitors of mitochondrial energy metabolism result in an indirect excitotoxic neuronal degeneration. We examined whether systemic administration of the mitochondrial toxin 3-nitroproprionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which also acts by an indirect excitotoxic mechanism, would produce alterations in the immunohistochemical pattern of KAT I. Our present investigations demonstrate that after 15 days of administration of 3-NP, an inhibitor of mitochondrial Complex II, the most severe depletion of KAT I occurred in the striatum; less severe depletion occurred in other brain areas investigated, following a striatum > hippocampus > temporal cortex gradient. The alterations induced by 15 days of 3-NP treatment were less conspicuous in 6-week-old (young) animals than in 3-month-old adults. In these adult animals, 3-NP induced necrotic cores in the striatum, characterized by destruction of neuronal and glial elements, similar to that seen in the histologic and neurochemical features of Huntington's disease. It appears that immunohistochemical depletion of KAT after administration of 3-NP to adult animals may contribute to the pathological processes that characterize Huntington's disease.     

A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury.

Various studies describe increased concentrations of transforming growth factor-beta (TGF-beta) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-beta after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-beta produced after an episode of cerebral ischemia by injecting a soluble TGF-beta type II receptor fused with the Fc region of a human immunoglobulin (TbetaRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-beta. Then, the authors tested its ability to reverse the effect of TGF-beta1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 microg/mL of TbetaRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-beta1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-beta1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-beta1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 microg of TbetaRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-beta1 mRNA. Furthermore, when added to the excitotoxin, TbetaRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TbetaRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-beta1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 +/- 9.5 versus 152.8 +/- 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-beta in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-beta1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.     

Repetitive intermittent hypoxia-ischemia and brain damage in neonatal rats

OBJECTIVE: To know the effect of brief-repetitive intermittent hypoxia-ischemia on the development of perinatal brain damage. STUDY DESIGN: Seven-day-old Wistar rats underwent ligation of the unilateral common carotid artery. The animals were allocated to three groups (n=12 in each group) and exposed to 8% oxygen as follows: group A: continuous exposure for 180 min; group B: continuous exposure for 90 min; and group C: 10 min of exposure repeated at 10-min intervals over a period of 180 min (total exposure time, 90 min). Seventy-two hours after exposure to hypoxia, the cerebral cortex was examined to assess the degree of neuronal necrosis and brain damage was classified into four grades of severity, 0-3. To evaluate the extent of brain damage, we used immunohistochemical staining with TIB-128 antibody, which reacts to MAC-1 antigen specific to microglia, and observed the glial reaction in the cerebral cortex, hippocampus, thalamus, and striatum. RESULTS: All the brain damage observed in groups A-C occurred on the side where the ligation was performed. The most severe damage was found in group A animals, of which seven showed significant neuronal necrosis, having a grade 2 or more advanced lesion. In group B, neuronal necrosis was modest, with only one animal having a grade 2 lesion. In group C, a significant neuronal necrosis was found in six animals despite having the same period of hypoxic exposure as those in group B. MAC-1 positive cells appeared in the cerebral cortex of histologically damaged animals and extended to the hippocampus, thalamus, and striatum in severely damaged animals from groups A, B, and C. CONCLUSION: Examination of the neonatal rat model suggested that repetitive and intermittent, rather than continuous hypoxia-ischemia, causes pronounced damage in the immature brain.     

Patterns of excitotoxin-induced brain lesions in the newborn rabbit: a neuropathological and MRI correlation

There is pressing need to employ new advances in structural MR brain imaging to better diagnose brain damage in newborn infants. Timely application of such technology will enable improved therapeutic interventions. Diffusion-weighted sequences are a sensitive marker of very early neuronal injury, the spatial pattern of which provides critical information regarding the underlying pathophysiology. We have modified our murine model of excitotoxic neonatal brain injury to the rabbit, an animal whose brain is larger and where the neuroanatomic organization of the subcortical white matter more closely resembles that of the human. Utilizing this rabbit model, we undertook an MRI/histopathologic correlation. We found that as with the mouse, there is a spatiotemporal selectivity to the pattern of brain injury, and that the period from postnatal day (P) 7 to P9 in rabbits corresponds to the time of maximum vulnerability of the brain to excitotoxic white matter damage, which neuropathologically simulates periventricular leukomalacia (PVL). We additionally noted that diffusion-weighted imaging provided the most sensitive means of detecting such lesions and that this method was sensitive to structural maturational changes accompanying the normal cortical ontogeny. Taken together, our findings suggest that this rabbit model of perinatal excitotoxic brain injury will be a valuable addition to experimental approaches to further our understanding of perinatal brain damage, that diffusion-weighted imaging will be an invaluable adjunct to the diagnosis of such injury, and that therapeutic strategies aimed at interrupting the evolution of PVL should include targeting the pathophysiologic cascade induced by excitotoxic neonatal brain injury.     

Differential sensitivity of murine astrocytes and neurons from different brain regions to injury

Different brain regions show differential vulnerability to ischemia in vivo. Despite this, little work has been done to compare vulnerability of brain cells isolated from different brain regions to injury. Relatively pure neuronal and astrocyte cultures were isolated from mouse cortex, hippocampus, and striatum. Astrocyte vulnerability to 6 h oxygen-glucose deprivation was greatest in striatum (81.8 +/- 4.6% cell death), intermediate in hippocampus (59.8 +/- 4.8%), and least in cortex (37.0 +/- 3.5%). In contrast neurons deprived of oxygen and glucose for 3 h showed greater injury to cortical neurons (71.1 +/- 5.2%) compared to striatal (39.0 +/- 3.1%) or hippocampal (39.0 +/- 5.3%) neurons. Astrocyte injury from glucose deprivation or H(2)O(2) exposure was significantly greater in cells from cortex than from striatum or hippocampus. Neuronal injury resulting from serum deprivation was greater in cortical neurons than in those from striatum or hippocampus, while excitotoxic neuronal injury was equivalent between regions. Antioxidant status and apoptosis-regulatory genes were measured to assess possible underlying differences. Glutathione was higher in astrocytes and neurons isolated from striatum than in those from hippocampus. Superoxide dismutase activity was significantly higher in striatal astrocytes, while glutathione peroxidase activity and superoxide did not differ by brain region. Bcl-x(L) was significantly higher in striatal astrocytes than in astrocytes from other brain regions and higher in striatal and hippocampal neurons than in cortical neurons. Both neurons and astrocytes isolated from different brain regions demonstrate distinct patterns of vulnerability when placed in primary culture. Antioxidant state and levels of expression of bcl-x(L) can in part account for the differential injury observed. This suggests that different protective strategies may have different efficacies depending on brain region.     

Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain

Cytokines are important intercellular messengers involved in neuron-glia interactions and in the microglial-astroglial crosstalk, modulating the glial response to brain injury and the lesion outcome. In this study, excitotoxic lesions were induced by the injection of N-methyl-D-aspartate in postnatal day 9 rats, and the cytokines interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumour necrosis factor alpha (TNFalpha) and transforming growth factor beta 1 (TGF-beta1) analysed by ELISA and/or immunohistochemistry. Moreover, cytokine-expressing glial cells were identified by means of double labelling with glial fibrillary acidic protein or tomato lectin binding. Our results show that both neurons and glia were capable of cytokine expression following different patterns in the excitotoxically damaged area vs. the nondegenerating surrounding grey matter (SGM). Excitotoxically damaged neurons showed upregulation of IL-6 and downregulation of TNFalpha and TGF-beta1 before they degenerated. Moreover, in the SGM, an increased expression of neuronal IL-6, TNFalpha and TGF-beta1 was observed. A subpopulation of microglial cells, located in the SGM and showing IL-1beta and TNFalpha expression, were the earliest glial cells producing cytokines, at 2-10 h postinjection. Later on, cytokine-positive glial cells were found within the excitotoxically damaged area and the adjacent white matter: some reactive astrocytes expressed TNFalpha and IL-6, and microglia/macrophages showed mild IL-1beta and TGF-beta1. Finally, the expression of all cytokines was observed in the glial scar. As discussed, this pattern of cytokine production suggests their implication in the evolution of excitotoxic neuronal damage and the associated glial response.     

Versican and brevican are expressed with distinct pathology in neonatal hypoxic-ischemic injury

The developing brain is uniquely susceptible to injury after exposure to hypoxia-ischemia (H-I). Lecticans are developmentally regulated in formative white matter and exert growth-inhibitory effects in several adult injury models, yet little is known regarding their role in neonatal H-I injury. The main objectives of this study were to examine the expression profiles of brevican and versican in rat using a standard H-I model and to determine whether altered expression was associated with distinct components of white and gray matter pathology. The H-I procedure in postnatal day 7 rats produced progressive injury limited to the ipsilateral hemisphere. Cresyl violet staining revealed severe cavitary infarctions at 14 and 21 days that were absent at 4 days. Cellular damage, as measured by glial fibrillary acidic protein and fractin immunoreactivity, occurred in cortical and subcortical gray matter at all end points. O4 sulfatide immunoreactivity was reduced in the external capsule, hippocampal fimbria, and corpus striatum at 4 days relative to that contralaterally, suggesting the loss of preoligodendrocytes. Brevican expression was reduced in the cortex and hippocampus at 4 days but was markedly elevated at later end points, localizing to regions of cellular damage both in and proximal to the lesion core. However, versican was reduced in the external capsule 4 days after H-I, a reduction that was sustained up to 21 days in white matter. These data demonstrate unique expression profiles for lecticans after neonatal H-I, suggesting brevican deposition is elevated in response to progressive gray matter injury, whereas diminished versican expression may be associated with deep cerebral white matter injury.