Glucose deprivation neuronal injury in cortical culture

Murine cortical cell cultures deprived of glucose for 6-8 h developed extensive neuronal degeneration, apparent both morphologically and by efflux of lactate dehydrogenase to the bathing medium. This neuronal damage could be substantially reduced by addition of D-2-amino-5-phosphonovalerate (D-APV), in a concentration-dependent (IC50 about 2 microM) and stereospecific (D-APV more potent than L-APV) fashion. A similar neuron-protective effect could also be obtained with several other NMDA antagonists, 2-amino-7-phosphonoheptanoate, phencyclidine, MK-801, ketamine, and (+)-SKF 10,047, as well as with the broad spectrum glutamine antagonist kynurenate. In contrast, little protection could be obtained with gamma-D-glutamylaminomethyl sulfonate and L-glutamate diethyl ester, compounds which have been reported to act primarily at non-NMDA receptors. These observations support the hypothesis that glucose deprivation-induced cortical neuronal injury is largely mediated by NMDA receptors, and suggest that cell culture methodology can be useful in the quantitative characterization of that injury.     

N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis." Copyright 1999 Academic Press.     

Oligomers of beta-amyloid peptide inhibit BDNF-induced arc expression in cultured cortical Neurons

The progressive memory loss observed in Alzheimer's disease (AD) is accompanied by an increase in the levels of amyloid-beta peptide (Abeta) and a block of synaptic plasticity. Both synaptic plasticity and memory require changes in the expression of synaptic proteins such as the activity-regulated cytoskeleton-associated protein, Arc (also termed Arg3.1). Using a model of synaptic plasticity in which BDNF increases Arc expression in cultured cortical neurons, we have found that an oligomeric form of Abeta strongly inhibits the BDNF-induced increase of Arc expression. Given that Abeta oligomers are likely to be involved in the synaptic dysfunction and cognitive impairment observed in amyloid depositing mouse models, we hypothesize that inhibition of Arc induction by BDNF contributes to the synaptic and memory deficits at early stages of AD.     

Topographical analysis of cortical neuronal loss associated with disseminated selective neuronal necrosis and infarction after repeated ischemia

A study was carried out of the distribution and density of the neurons remaining in the gerbil cerebral cortex following two 10-min periods of ischemia at either 3-, 5- or 48-h intervals. As the interval between the periods of ischemia increased, the ischemic injury was reduced from severe to milder infarction, and further from more to less intense disseminated selective neuronal necrosis. This model is suitable for studying the mechanisms of transition from selective neuronal necrosis to infarction at the threshold level of infarction.     

gamma-Aminobutyric acid-induced potentiation of cortical hemiplegia

A novel model of hemiplegia in young and aged rats is described. Osmotic minipumps were used to deliver a chronic (7 days), localized application of gamma-aminobutyric acid (GABA) (100 micrograms/microliter/h), to the somatomotor cortex of unrestrained rats. This resulted in an easily quantifiable, contralateral and reversible motor syndrome in both young and aged animals. In the young group, the motor deficit cleared over 5-day period, while in the aged animals it persisted for at least a 2-week period. Control animals treated with saline-filled minipumps did not develop a long-lasting motor deficit. The GABA-induced facilitation of hemiplegia due to small motor cortex lesions and the age effects on behavioral recovery of function are discussed. Cortical inhibitory mechanisms may play a role in debilitating syndromes such as stroke or post-epileptic paralysis.     

Copper activates TrkB in cortical neurons in a metalloproteinase-dependent manner

Copper (Cu) is an endogenous metal that is physiologically essential in the brain and that, like zinc (Zn), may be synaptically released in certain regions. Previously, we demonstrated that Zn activates TrkB in cultured cortical neurons in a metalloproteinase (MP)-dependent manner. To determine whether Cu has similar properties, we exposed cortical cultures for 15 min to various metals and performed Western blots to detect tyrosine-phosphorylated TrkB (p-Trk). Whereas Cd, Mn, Fe(II), and Fe(III) had no effect on the level of p-Trk, 10 microM of Cu in phosphate-containing (Hanks' balanced salt solution) or 10 nM in phosphate-lacking salt solution (control salt solution), increased levels of p-Trk. Cu also activated extracellular signal-regulated kinase 1/2 and Src tyrosine kinase, signaling molecules activated downstream of TrkB. Cu decreased levels of probrain-derived neurotrophic factor (pro-BDNF) in cells but increased levels of pro- and mature BDNF in the media. Addition of MP inhibitors completely blocked the Cu-induced increases in pro-BDNF and BDNF as well as TrkB activation, indicating that MP mediates most of the Cu effect. Furthermore, Cu increased the activity of matrix metalloproteinase 2 (MMP2) and MMP9 in cortical neurons. These findings indicate that, like Zn, Cu activates MPs, releases pro-BDNF from cells, and phosphorylates TrsB. Because Cu, like Zn, is released in certain brain areas with neuronal activity, metal-triggered TrkB activation may occur in both Cu- and Zn-containing synapses.     

A metabotropic glutamate receptor agonist does not mediate neuronal degeneration in cortical culture.

In light of the evidence that calcium plays a critical role in excitotoxic neuronal death, it has been speculated that the metabotropic glutamate receptor may also contribute to excitotoxic damage through the mobilization of Ca2+ from intracellular stores. In the present study we examined this possibility by studying the neurotoxicity of trans-1-amino-cyclopentyl-1,3-dicarboxylate (trans-ACPD), a selective agonist of the metabotropic glutamate receptor. Exposure of cortical neurons to 100 microM trans-ACPD substantially increased phosphoinositide hydrolysis and intraneuronal free calcium in the presence of CPP and CNQX. Despite the presence of functional metabotropic receptors on cultured neurons, however, exposure of cultures to as high as 1 mM trans-ACPD for 24 h failed to produce any morphological or chemical signs of neuronal damage. Furthermore, trans-ACPD did not potentiate submaximal neurotoxicity produced by other non-N-methyl-D-aspartate (NMDA) agonists, kainate and D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA).     

Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells

Previous studies have shown that BDNF promotes expression of SS. In earlier studies we demonstrated the stimulatory effect of locally produced VIP upon SS secretion. These facts led us to explore the peptidergic action of BDNF on VIP, and to determine if BDNF-induced SS might be mediated by the induction of VIP. Cultured fetal rat cerebrocortical cells were incubated with BDNF (50 ng/ml) and/or VIP (10(-11) M) for 2 and 5 days. In other experiments IgGs from BDNF or VIP antisera were also added. BDNF increased VIP and SS gene expression and peptide production. After 2 days of incubation with both BDNF and VIP the induction of SS mRNA was similar to that obtained with BDNF alone. However when the treatment was extended to 5 days the increase in SS mRNA was higher than that obtained with BDNF alone. This finding suggests the possibility that both factors acted synergistically. To define the potential role of VIP in the response of SS gene expression to BDNF, endogenous VIP was blocked with IgGs from VIP antiserum. Under these experimental conditions BDNF-induced SS decreased. Our study provides the first evidence that BDNF up-regulates VIP gene expression and concentration of the peptide. The involvement of VIP on BDNF-induced SS gene expression is also demonstrated.     

Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay

Measurement of lactate dehydrogenase (LDH) activity released to the extracellular bathing media has been found to be a simple yet quantitative method for assessing glutamate mediated central neuronal cell injury in cortical cell culture. Extracellular LDH is both chemically and biologically stable; the magnitude of LDH efflux in the cultures correlates in a linear fashion with the number of neurons damaged by glutamate exposure.     

Zn2+ entry produces oxidative neuronal necrosis in cortical cell cultures

Evidence has accumulated that Zn²⁺ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn²⁺ neurotoxicity. Inclusion of 30–300 μm Zn²⁺ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn²⁺ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn²⁺ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn²⁺-induced neuronal necrosis. Pretreatment with insulin or brain-derived neurotrophic factor increased the Zn²⁺-induced free radical injury. Kainate or AMPA facilitated Zn²⁺ entry and potentiated Zn²⁺ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn²⁺ neurotoxicity. These findings indicate that entry and accumulation of Zn²⁺ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.     

Tumor necrosis factor-alpha and interleukin-18 modulate neuronal cell fate in embryonic neural progenitor culture

Neural progenitor cells (NPCs) in developing and adult CNS are capable of giving rise to various neuronal and glial cell populations. Neurogenesis in the adult hippocampus has been found to be inhibited by a proinflammatory cytokine, interleukin-6 (IL-6), suggesting that activated microglia in the inflamed brain may control neurogenesis. Yet, little is known about the effect of microglia-derived factors on the cell fate of embryonic NPCs. In this study, we show that neurons with betaIII-tubulin immunoreactivity in the NPC culture were reduced by the condition media collected from microglia treated with endotoxin lipopolysaccharide (LPS/M-CM). Treatment with pentoxifylline (PTX), an inhibitor for tumor necrosis factor-alpha (TNF-alpha) secretion from LPS-activated microglia, blocked the reduction of betaIII-tubulin+ cells in NPC culture. Furthermore, treatment of NPCs with interleukin-18 (IL-18), a recently discovered proinflammatory cytokine, also decreased the number of betaIII-tubulin+ cells in a dose- and time-dependent manner. Surprisingly, we also observed that the remaining betaIII-tubulin+ cells in the LPS/M-CM-treated culture exhibited more branching neurites. Thus, the activated microglia-derived cytokines, TNF-alpha and IL-18, may either inhibit the neuronal differentiation or induce neuronal cell death in the NPC culture, whereas these cells may also produce factors to improve the neurite branching in the NPC culture.     

Laminar cortical necrosis in mitochondrial disorders

Objectives

Laminar cortical necrosis, defined as focal or diffuse necrosis of one or more cortical lamina, represents an increasingly recognized neuropathological endpoint of vascular, endocrine, immunologic, metabolic, or toxic conditions, of which mitochondrial disorders (MIDs) are the third most frequent after cerebral ischemia and hypoxia.

Aims

To investigate the prevalence of laminar cortical necrosis in MIDs, types of MIDs associated with laminar cortical necrosis, and the morphological characteristics on imaging and autopsy.

Methods

Medline literature review for the terms “laminar cortical necrosis”, “cortical signal change”, “mitochondrial” and all acronyms of syndromatic MIDs.

Results

Among 139 hits for “laminar cortical necrosis”, 10 articles fulfilled the inclusion criteria (7%). Among the ten hits five were case series and the other five single case reports. The syndromic MID most frequently associated with laminar cortical necrosis is the MELAS syndrome, but was also described in a single patient each with Leigh syndrome, mitochondrial depletion syndrome, and mitochondrial spinocerebellar ataxia. The morphological and pathohistological features of laminar cortical necrosis in MIDs were not at variance from those in non-mitochondrial disorders.

Conclusions

In MIDs laminar cortical necrosis represents the histopathological and imaging endpoint of a stroke-like lesion. Though laminar cortical necrosis may have a wide pathophysiological background the histological and imaging characteristics do not vary between the different underlying conditions.     

Paradoxical potentiation by low extracellular Ca2+ of acute chemical anoxic neuronal injury in cerebellar granule cell culture

Acute chemical anoxic injury was produced in primary cerebellar granule cell cultures incubated with iodoacetate (IAA) alone or IAA combined with potassium cyanide (KCN). Cytotoxicity was assessed using Trypan blue exclusion or LDH release. Four millimolars of KCN induced approx 30% neuron death at 3 h, whereas >50% cell death was produced by 0.2 mM IAA. No potentiation of cytotoxicity was observed by IAA+KCN. A total of 0.2 mM IAA produced an early major reduction of intracellular ATP prior to the onset of neuron injury or reduction in intracellular glutathione (GSH). Medium Na⁺ replacement by choline, K⁺, or methylglucamine protected against IAA-induced neuronal injury, reduced the rate of decline of intracellular ATP but had no effect on intracellular GSH. Some 80% neuronal survival was obtained when Na⁺ was deleted from the medium even after the intracellular ATP had been reduced to <10% of control. Removal of Ca²⁺ from the medium had no effect on control culture, Trypan blue exclusion, GSH, or ATP, but potentiated the onset and magnitude of IAA-induced cytotoxicity, ATP and GSH decline. Loading of granule cells with the Ca²⁺ chelator Fura-2 did not influence IAA-induced cytotoxicity in control or low Ca²⁺ media. Addition of 50 μM glutamate had a minimal cytotoxic effect over 3 h and the combined addition of 0.2 mM IAA plus 50 μM glutamate did not potentiate IAA-induced injury. The glutamate receptor antagonists,d-2-amino-5-phosphonovaleric acid (APV) or kynurenate did not block IAA-injuced injury in control medium but inhibited the potentiation of toxicity seen in the low Ca²⁺ medium. This study suggests the use of IAA as a chemical anoxic agent in cerebellar granule cell culture. The early, dose-dependent decline in ATP may be dissociated from GSH change. Acute IAA-induced injury is Na⁺/Cl^? dependent but paradoxically potentiated in low Ca²⁺ medium. The low Ca²⁺ potentiated component was sensitive to glutamate/NMDA receptor antagonists and associated with reduction of intracellular GSH.     

Nitric oxide mediates long-term potentiation in rat superior cervical ganglion

Long-term potentiation (LTP) of the nicotinic pathway of the superior cervical ganglion (SCG) is remarkably similar to that of the hippocampus which has been shown to involve nitric oxide (NO). We investigated a similar possible involvement of NO in the LTP of the SCG of the rat. Treatment of ganglia with the NO-synthase inhibitor N^G-nitro-l-arginine (l-NOARG, 10 μM) blocked LTP at the maintenance phase. © 1997 Elsevier Science B.V. All rights reserved.     

Excitotoxins in neuronal apoptosis and necrosis.

Neuronal loss is common to many neurodegenerative diseases. Although necrosis is a common histopathologic feature observed in neuropathologic conditions, evidence is increasing that apoptosis can significantly contribute to neuronal demise. The prevalence of either type of cell death, apoptosis or necrosis, and the relevance for the progression of disease is still unclear. The debate on the occurrence and prevalence of one or the other type of death in pathologic conditions such as stroke or neurotoxic injury may in part be resolved by the proposal that different types of cell death within a tissue reflect either partial or complete execution of a common death program. Apoptosis is an active process of cell destruction, characterized morphologically by cell shrinkage, chromatin aggregation with extensive genomic fragmentation, and nuclear pyknosis. In contrast, necrosis is characterized by cell swelling, linked to rapid energy loss, and generalized disruption of ionic and internal homeostasis. This swiftly leads to membrane lysis, release of intracellular constituents that evoke a local inflammatory reaction, edema, and injury to the surrounding tissue. During the past few years, our laboratories have studied the signals and mechanisms responsible for induction or prevention of apoptosis/necrosis in neuronal injury and this is the subject of this review.     

Dihydrokainate-sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l -serine-O-sulphate, but not by l -α-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20–24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 ± 9.8% in neuronal cultures (P < 0.001;n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05;n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 ± 4.9 μm , compared to 1.2 ± 0.3 μm in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 μm MK-801, 10 μm tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm ) inhibited the ⁴⁵calcium uptake stimulated by 30 μm N-methyl-d -aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced ≈ 25% inhibition of 30 μm NMDA-induced current in cortical neurons. Dihydrokainate (1 mm ) alone generated a small current (17% of the current produced by 30 μm NMDA) that was blocked by 30 μm 5,7-dichlorokynurenate and only weakly by 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.