Amyloid beta protein enhances the clearance of extracellular L-glutamate by cultured rat cortical astrocytes

To explore the impact of Alzheimer's disease amyloid beta protein (Abeta) on astrocyte functions, we investigated the effect of Abeta on glutamate clearance capacity of cultured rat cortical astrocytes. When L-glutamate (50-200 microM) was added to astrocyte cultures and incubated, the extracellular L-glutamate concentration declined with time. The time-dependent decline of extracellular L-glutamate was significantly faster in cultures treated with 10-20 microM Abeta for 24 h than in intact cultures, suggesting that Abeta enhances the L-glutamate clearance capacity of astrocytes. The effect of Abeta was not affected by antioxidants including catalase, propyl gallate or Trolox. Relatively long treatment time (8-48 h) was required for Abeta to exert this effect. Western blot analysis revealed that expression level of the glutamate transporter GLAST was increased by treatment with 10-20 microM Abeta for 8-48 h. These results suggest that Abeta upregulates a glutamate uptake system of astrocytes and enhances the clearance of extracellular L-glutamate.     

Direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the CA1 region of the rat hippocampus

Recent studies have demonstrated that astrocytes express a variety of ion channels and neurotransmitter receptors and can modulate the activity of neurons. Since a single astrocyte makes tight contacts with many neighboring neuronal cells, they can provide efficient and wide modulation of neuronal networks. Here, we provide direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the stratum radiatum of the rat hippocampus. Direct depolarization of a perineuronal astrocyte suppressed the excitatory postsynaptic currents in an adjacent interneuron and increased the paired-pulse ratio, indicating that perineuronal astrocytes have a suppressive effect on presynaptic elements. Moreover, perineuronal astrocyte activation modulated the directly induced firing pattern of the interneuron, with initial facilitation and subsequent suppression. Conversely, direct firing of the interneuron depolarized the membrane potential and reduced the input resistance of the perineuronal astrocyte. These results directly demonstrate the existence of bidirectional interactions between neurons and perineuronal astrocytes.     

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production.

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.     

Alpha(1)-adrenoceptor-mediated depolarization and beta-mediated hyperpolarization in cultured rat dorsal root ganglion neurones

The effect of excitotoxic injury on the production of monocyte chemoattractant protein-1 (MCP-1) was examined in rat cortico-striatal slice cultures. Treatment with 50 microM N-methyl-D-aspartate (NMDA) for 4 h, which caused severe damage in neurons, induced the production of MCP-1 in astrocytes. Production levels were markedly elevated immediately after the treatment, peaked at 4-8 h, and had decreased to nearly the basal level by 72 h. Since the treatment promoted the release of MCP-1 in the slice cultures, but not in the enriched astrocyte cultures, it is unlikely that NMDA directly acted on the astrocytes. These results suggest that information on neuronal injury induced by NMDA is transmitted to astrocytes to induce the production of MCP-1. Organotypic slice cultures are useful for investigating the inflammatory responses of astrocytes in the process of neuronal injury.     

Characterization of cortical astrocytes on materials of differing surface chemistry.

The behavior of cortical astrocytes was evaluated on a number of medically relevant materials of differing physicochemical properties. This study describes cell attachment, DNA synthesis, production of extracellular matrix (ECM) proteins, and neuronal interactions of perinatal rat astrocytes in vitro. The number of attached astrocytes initially differed among the materials, decreasing with increasing material hydrophobicity. In contrast, the rate of DNA synthesis increased with increasing material hydrophobicity. With the exception of only one material, astrocytes reached confluence by 12 days in culture on all the materials tested. Furthermore, the expression of characteristic ECM proteins and the fundamental ability of astrocytes to support neuronal attachment and growth was qualitatively identical between populations of astrocytes on different materials. The ability of astrocytes to colonize different surfaces initially was mediated via adsorbed serum proteins, as reducing the capacity of a model surface to adsorb proteins inhibited astrocyte colonization for up to 2 weeks in culture. We propose that astrocytes are relatively insensitive to differences in surface chemistries so long as the proteins necessary for cellular attachment are capable of adsorbing to the material to some extent. It seems likely that the ability of astrocytes to produce and remodel a matrix creates a surface environment that eventually becomes similar regardless of the surface chemistry of the underlying material.     

Angiotensinogen production by rat astroglial cells in vitro and in vivo

To investigate the production of angiotensinogen by the brain, primary cultures were prepared from the brains of one-day-old rats. Two to four weeks after plating, they were transferred to serum-free medium. The cultures, which contained approximately 15% neurons, 80% astroglia and 5% other types of cells, produced angiotensinogen at a steady rate for three to four days in serum-free medium. Cultures prepared from subcortical tissue produced more angiotensinogen than cultures prepared from cerebral cortical tissue. Angiotensinogen mRNA was also identified in those cultures. Forskolin treatment had no effect on angiotensinogen production. Astroglia-enriched cultures that contained no identifiable neurons also produced angiotensinogen and its mRNA. Astroglial cells from hypothalamus and thalamus produced more of both than astroglial cells from the cerebral cortex. In situ hybridization histochemistry on sections of the hypothalamus of adult male rats showed a diffuse distribution of cells containing angiotensinogen mRNA that was more consistent with a glial than a neuronal distribution. The data indicate that most if not all of the angiotensinogen in rat brain is produced by astrocytes.     

Lithium stimulates progenitor proliferation in cultured brain neurons

The number of neurons in the brain is controlled by production of new neurons and neuronal death. Neural progenitor proliferation in the developing and adult brain plays a prominent role in the production of new neurons. Here, we examined the effects of lithium, a mood-stabilizing drug, on neuronal proliferation in rat primary neuronal cultures. The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA was used to label proliferating cells. BrdU incorporation was detected by immunocytochemistry in cerebellar granule cells prepared from postnatal rats and cerebral cortical cultures prepared from embryonic rats. Quantification of BrdU incorporation into cultures was performed by counting BrdU-positive cells and BrdU-coupled enzyme-linked immunosorbent assay. Both methods revealed that lithium increased BrdU incorporation in cerebellar granule cells and cerebral cortical cultures. Most BrdU-positive cells colocalized with nestin, a neuroblast cell marker, in cerebral cortical cultures. Blockade of DNA replication by cytosine arabinoside almost completely abolished BrdU incorporation, suggesting that lithium-induced BrdU incorporation was mainly due to enhanced DNA replication. Glutamate, glucocorticoids and haloperidol were found to markedly reduce neural progenitor proliferation in cerebellar granule cells. The presence of lithium prevented the loss of proliferation induced by these agents. Lithium-induced neural progenitor proliferation in vitro suggests that similar effects might occur in vivo and this action could also be related to its clinical efficacy. Cultured brain neurons may provide a valuable model for studying the molecular mechanisms underlying lithium-induced up-regulation of neural proliferation.     

Differential neurite growth on astrocyte substrates: interspecies facilitation in green fluorescent protein-transfected rat and human neurons

In the present study, we used co-culture of astrocytes from one species with neurons from a different species to examine neuritic outgrowth. We include a focus on human cells. Three types of neuron were used, including rat hippocampal dentate granule cells, rat hypothalamic neurons and human cortical neurons. To visualize neuronal processes, neurons were either immunostained with GABA antiserum or transfected with the jellyfish green fluorescent protein gene. The entire axonal and dendritic fields of single neurons could be quantitatively analysed based on their strong green fluorescent protein label. Astrocytes were obtained from rat hippocampus or hypothalamus, chicken cortex, normal human cortex, human cortex lesion, and from the sclerotic human hippocampus after surgery for intractable temporal lobe epilepsy. In the absence of astrocytes, isolated neurons died within three to four days. In contrast, neurons from both rat and human brains survived and extended dendrites and axons on rat, chicken and human astrocytes or in their conditioned medium. Astrocytes from interspecies cultures were not only capable of enhancing the survival of neuron co-cultures, but neuronal neurite extension in some cases was even greater on heterospecific astrocytes than on homospecific astrocytes. To support the hypothesis that synaptogenesis of rat hippocampal neurons was accelerated by a substrate of human astrocytes, we used a functional assay based on time-lapse confocal laser or digital imaging of calcium responses to transmitter release; synaptic responses were found earlier when rat neurons were grown on rat or human astrocytes than in the absence of these astrocytes. These data indicate that rodent glial cells enhance human neurite extension, and that rat neurite outgrowth can be used as a type of bioassay for the neurite promoting capacity of different derivations of human glia.     

Differential neurite growth on astrocyte substrates: interspecies facilitation in green fluorescent protein-transfected rat and human neurons

In the present study, we used co-culture of astrocytes from one species with neurons from a different species to examine neuritic outgrowth. We include a focus on human cells. Three types of neuron were used, including rat hippocampal dentate granule cells, rat hypothalamic neurons and human cortical neurons. To visualize neuronal processes, neurons were either immunostained with GABA antiserum or transfected with the jellyfish green fluorescent protein gene. The entire axonal and dendritic fields of single neurons could be quantitatively analysed based on their strong green fluorescent protein label. Astrocytes were obtained from rat hippocampus or hypothalamus, chicken cortex, normal human cortex, human cortex lesion, and from the sclerotic human hippocampus after surgery for intractable temporal lobe epilepsy. In the absence of astrocytes, isolated neurons died within three to four days. In contrast, neurons from both rat and human brains survived and extended dendrites and axons on rat, chicken and human astrocytes or in their conditioned medium. Astrocytes from interspecies cultures were not only capable of enhancing the survival of neuron co-cultures, but neuronal neurite extension in some cases was even greater on heterospecific astrocytes than on homospecific astrocytes. To support the hypothesis that synaptogenesis of rat hippocampal neurons was accelerated by a substrate of human astrocytes, we used a functional assay based on time-lapse confocal laser or digital imaging of calcium responses to transmitter release; synaptic responses were found earlier when rat neurons were grown on rat or human astrocytes than in the absence of these astrocytes.These data indicate that rodent glial cells enhance human neurite extension, and that rat neurite outgrowth can be used as a type of bioassay for the neurite promoting capacity of different derivations of human glia.     

A role for astrocyte‐derived amyloid β peptides in the degeneration of neurons in an animal model of temporal lobe epilepsy

Kainic acid, an analogue of the excitatory neurotransmitter glutamate, can trigger seizures and neurotoxicity in the hippocampus and other limbic structures in a manner that mirrors the neuropathology of human temporal lobe epilepsy (TLE). However, the underlying mechanisms associated with the neurotoxicity remain unclear. Since amyloid‐β (Aβ) peptides, which are critical in the development of Alzheimer's disease, can mediate toxicity by activating glutamatergic NMDA receptors, it is likely that the enhanced glutamatergic transmission that renders hippocampal neurons vulnerable to kainic acid treatment may involve Aβ peptides. Thus, we seek to establish what role Aβ plays in kainic acid‐induced toxicity using in vivo and in vitro paradigms. Our results show that systemic injection of kainic acid to adult rats triggers seizures, gliosis and loss of hippocampal neurons, along with increased levels/processing of amyloid precursor protein (APP), resulting in the enhanced production of Aβ‐related peptides. The changes in APP levels/processing were evident primarily in activated astrocytes, implying a role for astrocytic Aβ in kainic acid‐induced toxicity. Accordingly, we showed that treating rat primary cultured astrocytes with kainic acid can lead to increased Aβ production/secretion without any compromise in cell viability. Additionally, we revealed that kainic acid reduces neuronal viability more in neuronal/astrocyte co‐cultures than in pure neuronal culture, and this is attenuated by precluding Aβ production. Collectively, these results indicate that increased production/secretion of Aβ‐related peptides from activated astrocytes can contribute to neurotoxicity in kainic acid‐treated rats. Since kainic acid administration can lead to neuropathological changes resembling TLE, it is likely that APP/Aβ peptides derived from astrocytes may have a role in TLE pathogenesis.     

Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice

Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO). IFN-gamma signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Abeta induced IFN-gamma production from co-culture of astrocytes and microglia, and IFN-gamma elicited tumor necrosis factor (TNF)-alpha secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-gamma and TNF-alpha enhanced Abeta production from APP-expressing astrocytes and cortical neurons. TNF-alpha directly stimulated beta-site APP-cleaving enzyme (BACE1) expression and enhanced beta-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-gamma and TNF-alpha activation of WT microglia suppressed Abeta degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-gamma and TNF-alpha enhance Abeta deposition through BACE1 expression and suppression of Abeta clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis.     

Pramipexole has astrocyte-mediated neuroprotective effects against lactacystin toxicity

Pramipexole, a dopamine D2/D3 receptor agonist used in the treatment of Parkinson's disease, has been reported to have neuroprotective potential. We investigated the effect of pramipexole against cell death induced by a proteasome inhibitor, lactacystin, using primary mecencephalic neuronal cultures and SH-SY5Y cells. In E14 rat primary mesencephalic cultures, the number of surviving tyrosine hydroxylase (TH)-positive neurons and microtubule associated protein 2 (MAP2)-positive neurons was decreased by exposure to 1-5 microM lactacystin in a dose-dependent manner. Pretreatment with 100 microM pramipexole rescued TH-positive neurons and MAP2-positive neurons from the toxicity of lactacystin. The protective effect of pramipexole was not selective for TH-positive dopaminergic neurons. However, the treatment with 100 microM pramipexole did not protect SH-SY5Y cells against lactacystin-induced cell toxicity and proteasome dysfunction. We hypothesized that the protective effect of pramipexole against the lactacystin-toxicity was not direct but a secondary effect mediated by astrocytes. Therefore, we investigated the efficacy of conditioned medium collected from mecencephalic astrocytes treated with pramipexole. The conditioned medium increased the viability of SH-SY5Y cells against the toxicity of lactacystin. Pramipexole increased the levels of brain derived neurotrophic factor (BDNF) in the conditioned medium of astrocyte cultures. These protective effects were not significantly inhibited by dopamine D2 or D3 receptor antagonists. We demonstrated that pramipexole had the protective effect against lactacystin toxicity, mediated by a neurotrophic effect of astrocyte-produced factors including BDNF.     

Effects of IL-6 secreted from astrocytes on the survival of dopaminergic neurons in lipopolysaccharide-induced inflammation

The role of astrocytes in microglia-induced neuronal death remains controversial. In this study, astrocytes and astrocyte-derived conditioned media (ACM) supported the survival of dopaminergic neurons, and the former was more effective than the latter. In the presence of astrocytes, low concentrations of LPS enhanced the survival of dopaminergic neurons, while high concentrations attenuated survival. LPS dramatically induced astrocytes to secrete IL-6 in a dose-dependent manner with no effect on secretion of GDNF. Neuron–astrocyte cultures had highest secretion of GDNF, followed by ACM-treated neuron-enriched cultures. After neuron–astrocyte cultures treated with IL-6-neutralizing antibody, both effects of the enhanced and attenuated survival of dopaminergic neurons were abolished. Our results indicate that astrocytes play a protective role in the LPS-induced damage of dopaminergic neurons in certain circumstances, and the interaction between astrocytes and dopaminergic neurons may enhance the protective effect of astrocytes. Suitable activation of astrocytes increases the protective effect while excessive activation attenuates it, and IL-6 might medicate this dual action. The underlying mechanisms related to the secretion of GDNF and proinflammatory factors warrant further investigation.     

Apolipoprotein-E modulates the cytotoxic effect of beta-amyloid on rat brain endothelium in an isoform-dependent specific manner

Several studies support the hypothesis that apolipoprotein-E (ApoE) acts as a pathological chaperone protein that promotes the beta-plated sheet conformation of beta-amyloid (Abeta) peptides into amyloid fibers. In vitro evidence is also available that ApoE inhibits the neurotoxic effect of Abeta in an allele-specific manner (E2 > or = E3 > E4). We have recently shown that Abeta peptides exert a time- and concentration-dependent toxic effect on rat neuromicrovascular endothelial cells (NECs), and this study aimed to ascertain whether ApoE isoforms are able to modulate this effect. ApoE2 and ApoE4 decreased and increased, respectively, the cytotoxic effect of Abeta(1-40) and Abeta(1-42) on NECs, as evaluated by their survival and viability rates. The toxic effect of both Abeta peptides and ApoE4 was associated with the rise in the necrosis rate of NECs within a 24-h incubation period. Moreover, ApoE2 prevented and ApoE4 magnified the inhibitory effect of Abeta on the capability of NECs cultured on Matrigel to form a capillary-like network. The opposite effects of ApoE isoforms could be due to their different interactions with the C-terminal domain of Abeta. ApoE2, at variance with ApoE4, is thought to form sodium dodecyl sulphate-stable complexes with Abeta and, as a consequence, it could block the interactions of the non-fibrillar Abeta peptide with the plasma membrane, Abeta peptide aggregation and the ensuing cytotoxicity. Collectively, our findings confirm the view that ApoE plays a relevant role in the pathogenesis of Alzheimer's disease.     

Astrocyte-derived estrogen enhances synapse formation and synaptic transmission between cultured neonatal rat cortical neurons

Recent in vitro studies have found that astrocytes exert powerful control over the number of neuronal synapses, leading us to consider why glia can exert this control and what the underlying mechanism(s) may be. To understand the potential possibility, we studied the formation of synapses and synaptic function in primary rat cortical neurons. We found that primary cultured neonatal rat cortical astrocytes modulate synaptogenesis and synaptic function through producing and secreting estradiol into culture medium. The concentration of estradiol produced by pure cultured astrocytes increased in correspondence with the days of culture and the number of proliferating astrocytes, which peaked at 266+/-22 ng/l around day 14 of culture. When astrocyte-conditioned medium (ACM) was added into pure cultured cortical neurons, the number of synapses formed between cortical neurons increased by nearly sixfold. The mean frequency and the amplitude of mini-postsynaptic currents (mPSCs) increased from 13+/-4 events/min and 20.5+/-2 pA to 73+/-16 events/min and 29.1+/-3 pA, respectively. In the meantime, the level of estrogen receptor-alpha (ER-alpha) expressed on neonatal rat cortical neurons was significantly up-regulated. Moreover, the effect of ACM on synaptic formation and transmission was blocked by tamoxifen (estrogen receptor antagonist) in culture. After the treatment of tamoxifen, the number of synapses on neurons decreased from 79+/-9 to 32+/-3. The mean amplitude and frequency of mPSCs were also dropped to 24.5+/-2 pA and 35+/-10/min, respectively. Unexpectedly, exogenic estradiol can mimic the effect of ACM on synaptic formation and transmission. Finally, to understand whether astrocyte-derived estradiol regulates the synaptic transmission via presynapse, the release of presynaptic vesicle from neuron was monitored by FM 4-64 assay. The results showed that when ACM or exogenic estradiol was added into neurons, the kinetics of vesicle release speed are similar to that of neuronal cultured with astrocytes, which were faster than that of just pure neuronal cultures. These observations suggest that estrogen synthesized and secreted by astrocytes can regulate synapse formation and synaptic transmission.     

Proinflammatory effects of M-CSF and A beta in hippocampal organotypic cultures

Macrophage colony stimulating factor (M-CSF) is a microglial activator expressed at increased levels in the brain in Alzheimer's disease. In monotypic microglial cultures, M-CSF strongly augments amyloid beta (Abeta) induced microglial production of proinflammatory cytokines and nitric oxide. However, this augmentation could be due to strong autocrine and paracrine effects in monotypic cultures. We used hippocampal organotypic cultures to test M-CSF/Abeta augmentation in a system modeling intact brain. Combined M-CSF/Abeta treatment increased interleukin-1 (IL-1) and macrophage inflammatory protein 1-alpha expression by microglia, whereas inducible nitric oxide synthase (iNOS) expression was localized primarily to astroglia. Induction of cytokines and iNOS was also observed after lipopolysaccharide treatment of organotypic hippocampal cultures, but iNOS expression was localized mainly to microglia rather than astrocytes. Treatment with M-CSF/Abeta did not result in neuronal death. These results demonstrate that combined M-CSF/Abeta treatment results in a strong inflammatory response in the organotypic environment without inducing neurotoxicity.     

Specific uptake of Abeta1-40 in rat brain occurs in astrocyte,but not in microglia

In the brain of a patient with Alzheimer's disease, beta amyloid peptide (Abeta) is thought to be taken up by glial cells such as astrocyte and microglia to be degraded. However, it is unclear whether the Abeta is absorbed by astrocyte or microglia. The purpose of our study is to determine which type of glial cell, astrocyte or microglia, can take up Abeta. Beta amyloid 1-40 (Abeta1-40) was directly infused into the frontal cortex or hippocampus for 14 days. Dual-labeling immunohistochemistry for Abeta1-40 with an astrocytic (GFAP) or microglial (CD11b) marker was performed to examine co-localization of Abeta1-40 and glial markers. In the Abeta1-40 infused site, immunoreactivity of Abeta1-40 was observed only in astrocytes, not in microglia. In addition, Abeta40-1, a reverse peptide of Abeta1-40, was not taken up by astrocytes. These results suggested that the astrocyte-specific uptake of Abeta occurred in the rat brain.     

Neuronal or glial expression of human apolipoprotein e4 affects parenchymal and vascular amyloid pathology differentially in different brain regions of double- and triple-transgenic mice

Apolipoprotein E4 (ApoE4) is associated with Alzheimer's disease by unknown mechanisms. We generated six transgenic mice strains expressing human ApoE4 in combination with mutant amyloid precursor protein (APP) and mutant presenilin-1 (PS1) in single-, double-, or triple-transgenic combinations. Diffuse, but not dense, amyloid plaque-load in subiculum and cortex was increased by neuronal but not glial ApoE4 in old (15 months) double-transgenic mice, whereas both diffuse and dense plaques formed in thalamus in both genotypes. Neuronal and glial ApoE4 promoted cerebral amyloid angiopathy as extensively as mutant PS1 but with pronounced regional differences: cortical angiopathy was induced by neuronal ApoE4 while thalamic angiopathy was again independent of ApoE4 source. Angiopathy correlated more strongly with soluble Abeta40 and Abeta42 levels in cortex than in thalamus throughout the six genotypes. Neither neuronal nor glial ApoE4 affected APP proteolytic processing, as opposed to mutant PS1. Neuronal ApoE4 increased soluble amyloid levels more than glial ApoE4, but the Abeta42/40 ratios were similar, although significantly higher than in single APP transgenic mice. We conclude that although the cellular origin of ApoE4 differentially affects regional amyloid pathology, ApoE4 acts on the disposition of amyloid peptides downstream from their excision from APP but without induction of tauopathy.     

Astrocytes are more resistant to focal cerebral ischemia than neurons and die by a delayed necrosis

Several recent reports proposed that astrocyte death might precede neuronal demise after focal ischemia, contrary to the conventional view that astrocytes are more resistant to injury than neurons. Interestingly, there are findings supporting each of these opposing views. To clarify these controversies, we assessed astrocyte viability after 2-h middle cerebral artery occlusion in mice. In contrast to neighboring neurons, astrocytes were alive and contained glycogen across the ischemic area 6 h after reperfusion, and at the expanding outer border of the infarct at later time points. These glycogen-positive astrocytes had intact plasma membranes. Astrocytes lost plasmalemma integrity much later than neurons: 19 ± 22 (mean ± standard deviation), 58 ± 14 and 69 ± 3% of astrocytes in the perifocal region became permeable to propidium iodide (PI) at 6, 24, 72 h after ischemia, respectively, in contrast to 81 ± 2, 96 ± 3, 97 ± 2% of neurons. Although more astrocytes in the cortical and subcortical core regions were PI-positive, their numbers were considerably less than those of neurons. Lysosomal rupture (monitored by deoxyribonuclease II immunoreactivity) followed a similar time course. Cytochrome-c immunohistochemistry showed that astrocytes maintained mitochondrial integrity longer than neurons. EM confirmed that astrocyte ultrastructure including mitochondria and lysosomes disintegrated much later than that of neurons. We also found that astrocytes died by a delayed necrosis without significantly activating apoptotic mechanisms although they rapidly swelled at the onset of ischemia.