Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischemia

The present study was designed to investigate the possible neuroprotective activity of ginseng roots in 5-min ischemic gerbils using a step-down passive avoidance task and subsequent neuron and synapse counts in the hippocampal CA1 region. The following drugs were administered for 7 days before the induced ischemia: red ginseng powder (RGP), crude ginseng saponin (CGS), crude ginseng non-saponin (CGNS), and pure ginsenosides Rb₁, Rg₁ and Ro. Oral administration of RGP significantly prevented the ischemia-induced decrease in response latency, as determined by the passive avoidance test, and rescued a significant number of ischemic hippocampal CA1 pyramidal neurons in a dose-dependent manner. Intraperitoneal injections of CGS exhibited a similar neuroprotective effect. CGNS had a significant but less potent protective effect against impaired passive avoidance task and degeneration of hippocampal CA1 neurons. Ginsenoside Rb₁ significantly prolonged the response latency of ischemic gerbils and rescued a significant number of ischemic CA1 pyramidal neurons, whereas ginisenosides Rg₁ and Ro were ineffective. Postischemic treatment with RGP, CGS or ginsenoside Rb₁ was ineffective. The neuroprotective activities of RGP, CGS and ginsenoside Rb₁ were confirmed by electron microscopy counts of synapses in individual strata of the CA1 field of ischemic gerbils pretreated with the drugs. These findings suggest that RGP and CGS are effective in the prevention of delayed neuronal death, and that ginsenoside Rb₁ is one of the neuroprotective molecules within ginseng root. Received: 22 May 1995 / Revised, accepted: 14 August 1995     

The Glutamate Transport Inhibitor L-trans-pyrrolidine-2,4-dicarboxylate Indirectly Evokes NMDA Receptor Mediated Neurotoxicity in Rat Cortical Cultures

Because of the well-documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L-trans-pyrrolidine-2,4- dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20-24 h, PDC was neurotoxic in conventional astrocyte-rich cortical cultures, with an EC₅₀ in these cultures of 320 ± 157 μM. In astrocyte-poor cultures, an EC₅₀ for PDC of 50 ± 5 μM was determined. The neurotoxicity of PDC in both astrocyte-rich and astrocyte-poor cultures was blocked by the NMDA antagonist MK-801, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre-loading cells with the non-metabolizable analogue of glutamate, [³H]-D-aspartate, first we demonstrated that PDC caused significant efflux of [³H]-D-aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC₅₀ of 18 ± 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC-evoked [³H]-D-aspartate release. PDC-evoked release of [³H]-D-aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate-pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter-dependent accumulation of glutamate in the extracellular space.     

Effects of ginsenosides on Ca channels and membrane capacitance in rat adrenal chromaffin cells

We investigated the effects of ginseng total saponins (GTS) and five ginsenosides on voltage-dependent Ca²⁺ channels and membrane capacitance using rat adrenal chromaffin cells. In this study, cells were voltage-clamped in a whole-cell recording mode and a perforated patch-clamp technique was used. The inward Ca²⁺ currents (I_Ca) was elicited by depolarization and the change in cell membrane capacitance (ΔC_m) was monitored. The application of GTS (100 μg/ml) induced rapid and reversible inhibition of the Ca²⁺ current by 38.8 ± 3.6% (n = 16). To identify the particular single component that seems to be responsible for Ca²⁺ current inhibition, the effects of five ginsenosides (ginsenoside Rb₁, Rc, Re, Rf, and Rg₁) on the Ca²⁺ current were examined. The inhibitions to the Ca²⁺ current by Rb₁, Rc, Re, Rf, and Rg₁ were 15.3 ± 2.2% (n = 5); 36.9 ± 2.4% (n = 7); 28.1 ± 1.9% (n = 12); 19.0 ± 2.5% (n = 10); and 16.3 ± 1.6% (n = 15), respectively. The order of inhibitory potency (100 μM) was Rc > Re > Rf > Rg₁ > Rb₁. A software based phase detector technique was used to monitor membrane capacitance change (ΔC_m). The application of GTS (100 μg/ml) induced inhibitory effects on ΔC_m by 60.8 ± 9.7% (n = 10). The inhibitions of membrane capacitance by Rb₁, Rc, Re, Rf, and Rg₁ were 35.3 ± 5.5% (n = 7); 41.8 ± 7.0% (n = 8); 40.5 ± 5.9% (n = 9); 51.2 ± 7.6% (n = 9); and 35.9 ± 5.1% (n = 10), respectively. The inhibitory potencies of the ginsenosides on ΔC_m were Rf > Rc > Re > Rg₁ > Rb₁. Therefore, we found that GTS and ginsenosides exerted inhibitory effects on both Ca²⁺ currents and ΔC_m in rat adrenal chromaffin cells. These results suggest that ginseng saponins regulate catecholamine secretion from adrenal chromaffin cells and this regulation could be the cellular basis of antistress effects induced by ginseng.     

Protection in Glutamate-Induced Neurotoxicity by Imidazoline Receptor Agonist Moxonidine

In the present study we investigated the effects of mixed imidazoline-1 and α₂-adrenoceptor agonist, moxonidine, in glutamate-induced neurotoxicity in frontal cortical cell cultures of rat pups by dye exclusion test. Also, phosphorylated p38 mitogen activated protein kinases (p-p38 MAPK) levels were determined from rat frontal cortical tissue homogenates by two dimensional gel electrophoresis and semidry western blotting. Glutamate at a concentration of 10^?6 M was found neurotoxic when applied for 16 hr in cell cultures. Dead cell mean scores were 12.8 ± 0.5 for control and 52.3 ± 4.8 for glutamate (p < .001). On the other hand, p-p38 MAPK levels start to increase at a glutamate concentration of 10^?7 M for 20 min application. Moxonidine was found to have an U-shape neuroprotective effect in glutamate-induced neurotoxicity in neuronal cell culture experiments. Even though moxonidine did not induce neurotoxicity alone between the doses of 10^?8 to 10^?4 M concentrations in cell culture series, it caused the reduction of glutamate-induced dead cell population 23.07 ± 3.6% in 10^?6 M and 26.7 ± 2.1% in 10^?5 M concentrations (p <.001 for both, in respect to control values). The protective effect of moxonidine was confirmed in 10^?8 and 10^?7 M, but not in higher concentrations in glutamate neurotoxicity in gel electrophoresis and western blotting of p-p38 MAPK levels. In addition to other studies that revealed an antihypertensive feature of moxonidine, we demonstrated a possible partial neuroprotective role in lower doses for it in glutamate-mediated neurotoxicity model.     

Ginsenosides Rb1 and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration. J Neurosci Res 53:426-432.

Kim YC, Kim SR, Markelonis GJ, Oh TH (1998): Ginsenosides Rb₁ and Rg₃ protect cultured rat cortical cells from glutamate-induced neurodegeneration. J Neurosci Res 53:426–432. On page 427 of the article referenced above, under the heading Assessment of Neurotoxicity in the Materials and Methods section, the formula given for the assessment of percentage cell viability was printed incorrectly. The formula is correctly stated in a footnote to Table 1 on page 429. The correct formula appears below: 100 × (OD of glutamate + ginsenoside-treated – OD of glutamate-treated)/(OD of control – OD of glutamate-treated). The publisher regrets this error.     

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.     

Ginsenosides protect striatal neurons in a cellular model of Huntington's disease

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), is a widely used herbal medicine. Ginsenosides, the active ingredients of ginseng, are the main components responsible for many beneficial actions of ginseng. In the present study, we tested 10 different ginsenosides in the previously developed in vitro Huntington's disease (HD) assay with primary medium spiny striatal neuronal cultures (MSN) from the YAC128 HD mouse model. We found that nanomolar concentrations of ginsenoside Rb1 and Rc effectively protected YAC128 medium spiny neurons from glutamate‐induced apoptosis and that Rg5 was protective at micromolar concentration. The other seven ginsenosides tested were not effective or exerted toxic effects in MSN cultures. From further experiments, we suggested that neuroprotective effects of ginsenosides Rb1, Rc, and Rg5 could correlate with their ability to inhibit glutamate‐induced Ca²⁺ responses in cultured MSN. From these results we concluded that ginsenosides Rb1, Rc, and Rg5 offer a potential therapeutic choice for the treatment of HD and possibly other neurodegenerative disorders. © 2009 Wiley‐Liss, Inc.     

Cholecystokinin-induced protection of cultured cortical neurons against glutamate neurotoxicity

The effects of cholecystokinin (CCK) on glutamate-induced neurotoxicity were examined using cultured rat cortical neurons. Brief exposure of glutamate followed by an incubation with normal solution for more than 60 min reduced cell viability by 60–70%, compared with control values. Glutamate-induced neurotoxicity was significantly inhibited by MK-801 and ketamine, which are non-competitive blockers of N-methyl-d-aspartate (NMDA) receptors. Octapeptide CCK-8S and CCK-related decapeptide ceruletide at concentrations of 10⁻⁹−10⁻⁷ M dose-dependently reduced glutamate-induced neurotoxicity. A desulfated analog CCK-8NS, which acts selectively as an antagonist of CCK_B receptors, also reduced glutamate neurotoxicity. The neuroprotective effects of CCK were antagonized by L-365260, a CCK_B receptor antagonist, but not by L-364718, a CCK_A receptor antagonist. These results suggest that CCK protects cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via CCK_B receptors.     

Arctigenin protects cultured cortical neurons from glutamate-induced neurodegeneration by binding to kainate receptor

We previously reported that arctigenin, a lignan isolated from the bark of Torreya nucifera, showed significant neuroprotective activity against glutamate-induced toxicity in primary cultured rat cortical cells. In this study, the mode of action of arctigenin was investigated using primary cultures of rat cortical cells as an in vitro system. Arctigenin significantly attenuated glutamate-induced neurotoxicity when added prior to or after an excitotoxic glutamate challenge. The lignan protected cultured neuronal cells more selectively from neurotoxicity induced by kainic acid than by N-methyl-D-aspartate. The binding of [(3)H]-kainate to its receptors was significantly inhibited by arctigenin in a competitive manner. Furthermore, arctigenin directly scavenged free radicals generated by excess glutamate and successfully reduced the level of cellular peroxide in cultured neurons. These results suggest that arctigenin exerted significant neuroprotective effects on glutamate-injured primary cultures of rat cortical cells by directly binding to kainic acid receptors and partly scavenging of free radicals.     

Neuroprotective action of donepezil mediated by neuronal nicotinic receptors

AChE inhibitors used in the treatment in Alzheimer’s disease such as donepezil are effective in preventing glutamate neurotoxicity. In primary cultures of the cortical neurons, donepezil prevents glutamate neurotoxicity when the drug is applied 8–24 hr prior to glutamate exposure. Neuroprotective effect of donepezil is antagonized by mecamylamine, dihydro‐β‐erythoridine and metyllcaconitine. Prolonged (more than 4 days) exposure of the cultures to donepezil induces an increase in the nicotine‐induced Ca²⁺ influx and number of neurons expressing α4 and α7 subunits of nicotinic receptors. Donepezil also prevents apoptotic neuronal death induced by glutamate. Inhibitors for non‐receptor type tyrosine kinase, Fyn and janus‐activated kinase 2, suppress the neuroprotective effect of donepezil. Neuroprotective effect of donepezil is also suppressed by a phosphatidylinositol 3‐kinase (PI3K) inhibitor. Phosphorylation level of Akt, an effector of PI3K, and the expression level of Bcl‐2 increase with donepezil. These results suggest that donepezil prevents glutamate neurotoxicity through α4‐ and α7‐nicotinic acetylcholine receptors (nAChRs), followed by activation of PI3‐Akt pathway but independent from activation of ion channels associated with nAChRs. α4‐ and α7‐nAChRs may play an important role in promoting survival of cortical neurons under oxidative stress caused by excitotoxicity.     

Neurotoxicity screening of (illicit) drugs using novel methods for analysis of microelectrode array (MEA) recordings

Annual prevalence of the use of common illicit drugs and new psychoactive substances (NPS) is high, despite the often limited knowledge on the health risks of these substances. Recently, cortical cultures grown on multi-well microelectrode arrays (mwMEAs) have been used for neurotoxicity screening of chemicals, pharmaceuticals, and toxins with a high sensitivity and specificity. However, the use of mwMEAs to investigate the effects of illicit drugs on neuronal activity is largely unexplored.We therefore first characterised the cortical cultures using immunocytochemistry and show the presence of astrocytes, glutamatergic and GABAergic neurons. Neuronal activity is concentration-dependently affected following exposure to six neurotransmitters (glutamate, GABA, serotonin, dopamine, acetylcholine and nicotine). Most neurotransmitters inhibit neuronal activity, although glutamate and acetylcholine transiently increase activity at specific concentrations. These transient effects are not detected when activity is determined during the entire 30 min exposure window, potentially resulting in false-negative results. As expected, exposure to the GABA_A-receptor antagonist bicuculline increases neuronal activity. Exposure to a positive allosteric modulator of the GABA_A-receptor (diazepam) or to glutamate receptor antagonists (CNQX and MK-801) reduces neuronal activity. Further, we demonstrate that exposure to common drugs (3,4-methylenedioxymethamphetamine (MDMA) and amphetamine) and NPS (1-(3-chlorophenyl)piperazine (mCPP), 4-fluoroamphetamine (4-FA) and methoxetamine (MXE)) decreases neuronal activity. MXE most potently inhibits neuronal activity with an IC₅₀ of 0.5 μM, whereas 4-FA is least potent with an IC₅₀ of 113 μM.Our data demonstrate the importance of analysing neuronal activity within different time windows during exposure to prevent false-negative results. We also show that cortical cultures grown on mwMEAs can successfully be applied to investigate the effects of different (illicit) drugs on neuronal activity. Compared to investigating multiple single endpoints for neurotoxicity or neuromodulation, such as receptor activation or calcium channel function, mwMEAs can provide information on integrated aspects of drug-induced neurotoxicity more rapidly. Therefore, this approach could contribute to a faster insight in possible health risks and shorten the regulation process.     

Involvement of platelet-activating factor (PAF) in glutamate neurotoxicity in rat neuronal cultures

To clarify the role of platelet-activating factor (PAF) in glutamate neurotoxicity, in vitro experiments using primary neuronal cultures were performed. The anti-PAF immunoglobulin-G (aPAF-IgG) and the three PAF receptor antagonists (BN52021, CV6209, and E5880) were tested for their neuroprotective activity in primary neuronal cultures isolated from embryonic rat cerebral cortex. The cultured neurons were exposed to glutamate (1 mM) for 60 min. Twenty-four hours after this exposure, aPAF-IgG demonstrated evidence of protective effects against neuronal damage in a dose-dependent manner. Protective effects also were observed in cultures treated with the three PAF antagonists (P<0.05 at 1 μg/ml aPAF-IgG, P<0.01 at 100 μM BN52021, P<0.05 at 10 nM CV6209 and P<0.01 at 10 nM E5880). The Fura-2 assay was used to estimate whether low dosages of exogenous PAF affect cultured neurons. The cultured neurons were loaded with Fura-2/AM. After preincubation for 120 min, the Fura-2-loaded neurons were exposed to various concentrations of PAF for 60 min. By measuring the fluorescent intensity of the medium as representing the amount of Fura-2 released from damaged neurons, we detected an increased release of Fura-2, even at low doses of PAF (P<0.01 at 10 nM PAF). We further studied PAF production by neurons in response to glutamate. The level of PAF measured in the medium exposed to glutamate was significantly higher than the level in the medium unexposed to glutamate (P<0.05). Our results suggest an important role of PAF in glutamate neurotoxicity. © 1997 Elsevier Science B.V. All rights reserved.     

Selective Blockade of the mGluR1 Receptor Reduces Traumatic Neuronal Injury in Vitro and Improves Outcome after Brain Trauma

The effects of selective blockade of group I metabotropic glutamate receptor subtype 1 (mGluR1) on neuronal cell survival and post-traumatic recovery was examined using rat in vitro and in vivo trauma models. The selective mGluR1 antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), and (S)-(+)-α-amino-4-carboxy-2-methylbezeneacetic acid (LY367385) provided significant neuroprotection in rat cortical neuronal cultures subjected to mechanical injury, in both pretreatment or posttreatment paradigms. Administration of the antagonists also attenuated glutamate-induced neuronal cell death in the cultures. Coapplication of these antagonists with the N-methyl- -aspartate (NMDA) receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) had additive neuroprotective effects in glutamate injured cultures. Intracerebroventricular administration of AIDA to rats markedly improved recovery from motor dysfunction after lateral fluid percussion induced traumatic brain injury (TBI). Treatment with mGluR1 antagonists also significantly reduced lesion volumes in rats after TBI, as evaluated by MRI. It appears that these compounds mediate their neuroprotective effect through an mGluR1 antagonist action, as demonstrated by inhibition of agonist induced phosphoinositide hydrolysis in our in vitro system. Moreover, AIDA, CPCCOEt, and LY367385, at concentrations shown to be neuroprotective, had no significant effects on the steady state NMDA evoked whole cell current. Taken together, these data suggest that modulation of mGluR1 activity may have substantial therapeutic potential in brain injury.     

Activation of Class II or III Metabotropic Glutamate Receptors Protects Cultured Cortical Neurons Against Excitotoxic Degeneration

Trans-1-aminocyclopentane-1,3-dicarboxylic acid, a mixed agonist of all metabotropic glutamate receptor (mGluR) subtypes, is known to produce either neurotoxic or neuroprotective effects. We have therefore hypothesized that individual mGluR subtypes differentially affect neurodegenerative processes. Selective agonists of subtypes which belong to mGluR class II or III, such as (2_s, 1′_R,2′_R,3′_R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV) (specific for subtypes mGluR2 or 3) or L-2-amino-4-phosphonobutanoate and L-serine-O-phosphate (specific for subtypes mGluR4, 6 or 7), were highly potent and efficacious in protecting cultured cortical neurons against toxicity induced by either a transient exposure to N-methyl-D-aspartate (NMDA) or a prolonged exposure to kainate. In contrast, agonists that preferentially activate class I mGluR subtypes (mGluR1 or 5), such as quisqualate or trans-azetidine-2,3-dicarboxylic acid, were inactive. DCG-IV was still neuroprotective when applied to cultures after the toxic pulse with NMDA. This delayed rescue effect was associated with a reduction in the release of endogenous glutamate, a process that contributes to the maturation of neuronal damage. We conclude that agonists of class II or III mGluRs are of potential interest in the experimental therapy of acute or chronic neurodegenerative disorders.     

Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α7-neuronal receptors and neuronal CNS receptors

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 μM) for 8–24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. α-bungarotoxin (α-BTX) and methyllycaconitine (MLA), both of which are α₇-neuronal receptor antagonists, and dihydro-β-erythroidine (DHβE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via α₇-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca²⁺ influx.     

Role of autophagy inhibitors and inducers in modulating the toxicity of trimethyltin in neuronal cell cultures

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Earlier ultrastructural studies indicated the formation of autophagic vacuoles in neurons after TMT intoxication. However, no evaluation has been attempted to determine the role of the autophagic pathway in TMT neurotoxicity. To assess the contribution of autophagy to TMT-induced neuronal cell death, we checked the vulnerability of neuronal cultures to TMT after activation or inhibition of autophagy. Our results show that autophagy inhibitors (3-methyladenine and l-asparagine) greatly enhanced TMT neurotoxicity. Conversely, known activators of autophagy, such as lithium and rapamycin, displayed neuroprotection against this toxic compound. Due to its diverse targets, the action of lithium was complex. When lithium was administered according to a chronic treatment protocol (6?days pretreatment) it was able to rescue both hippocampal and cortical neurons from TMT (or from glutamate toxicity used as reference). This effect was accompanied by an increased phosphorylation of glycogen synthase kinase 3 which is a known target for lithium neuroprotection. If the pre-incubation time was reduced to 2?h (acute treatment protocol), lithium was still able to counteract TMT toxicity in hippocampal but not in cortical neurons. The neuroprotective effect of lithium acutely administered against TMT in hippocampal neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. These data indicate that TMT neurotoxicity can be dramatically modified, at least in vitro, by lithium addition which seems to act through different mechanisms if acutely or chronically administered.     

Effects of TRH and its analogues on primary cortical neuronal cell damage induced by various excitotoxic, necrotic and apoptotic agents

The tripeptide thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) has been shown to possess neuroprotective activity in in vitro and in vivo models. Since its potential utility is limited by relatively rapid metabolism, metabolically stabilized analogues have been constructed. In the present study we investigated the influence of TRH and its three stable analogues: Montirelin (MON, CG-3703), RGH-2202 (L-6-keto-piperidine-2carbonyl-l-leucyl-l-prolinamide) and Z-TRH (N-carbobenzyloxy-pGlutamyl-Histydyl-Proline) in various models of mouse cortical neuronal cell injury. Twenty four hour pre-treatment with TRH and its analogues in low micromolar concentrations attenuated the neuronal cell death evoked by excitatory amino acids (EAAs: glutamate, NMDA, kainate, quisqualate) and hydrogen peroxide. All the peptides showed neuroprotective action on staurosporine (St)-evoked apoptotic neuronal cell death, but this effect was caspase-3 independent. Interestingly, in mixed neuronal-glial cell preparations only MON decreased St- and glutamate-evoked neurotoxicity. None of the peptides inhibited the doxorubicin- and lactacystin-induced neuronal cortical cell death, agents acting via activation of death receptor (FAS) or inhibition of proteasome function, respectively. Furthermore, we found that neither inhibitors of PI3-K (wortmannin, LY 294002) nor MAPK/ERK1/2 (PD 098059, U 0126) were able to inhibit neuroprotective properties of TRH and MON in St model of apoptosis. The protection mediated by TRH and MON it that model was also not connected with influence of peptides on the pro-apoptotic GSK-3β and JNK protein kinase expression and activity. Further studies showed that calpains, calcium-activated proteases were induced by Glu, but not by St in cortical neurons. Moreover, the Glu-evoked increase in spectrin alpha II cleavage product induced by calpains was blocked by TRH. The obtained data showed that the potency of TRH and its analogues in inhibiting EAAs- and H₂O₂-induced neuronal cell death from the highest to lowest activity was: MON > TRH > Z-TRH > RHG. Interestingly, all peptides were active against St-induced apoptosis, however, on concentration basis MON was far more potent than the other peptides. None of the peptides inhibited Dox- and LC-evoked apoptotic cell death. Additionally, the data exclude potential role of pro-survival (PI3-K/Akt and MAPK/ERK1/2) and pro-apoptotic (GSK-3β and JNK) pathways in neuroprotective effects of TRH and its analogues on St-induced neuronal apoptosis. Moreover, the results point to involvement of the inhibition of calpains in the TRH neuroprotective effect in Glu model of neuronal cell death.     

Prostaglandin E2 protects cultured cortical neurons against receptor-mediated glutamate cytotoxicity

The effects of prostaglandin (PG) E₂ on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to either glutamate or (NMDA) then incubated with normal medium for 1 h. Similar cytotoxicity was observed with the brief application of ionomycin, a calcium ionophore, and S-nitrosocysteine, a nitric oxide (NO)-generating agent. PGE₂ at concentrations of 0.01–1 μM dose-dependently ameliorated the glutamate-induced cytotoxicity. PGE₁, butaprost, an EP₂ receptor agonist, and 8-bromo-cAMP were also effective in protecting cultures against glutamate cytotoxicity. By contrast, neither 17-phenyl-ω-trinor-PGE₂, an EP₁ receptor agonist, nor M & B 28767, an EP₃ receptor agonist, affected glutamate-induced cytotoxicity. NMDA-induced cytotoxicity was ameliorated by PGE₂, butaprost, MK-801, , a NO synthase inhibitor, and hemoglobin, which binds NO. These agents excluding MK-801 ameliorated the ionomycin-induced cytotoxicity. The cytotoxicity induced by S-nitrosocysteine was prevented only by hemoglobin but not by the other agents including PGE₂. These findings indicate that PGE₂ protects cultured cortical neurons against NMDA receptor-mediated glutamate neurotoxicity via EP₂ receptors. EP₂ receptor stimulation may suppress a step in NO formation triggered by Ca²⁺-influx through NMDA receptors.     

Neuroprotective effects of gangliosides may involve inhibition of nitric oxide synthase

Gangliosides are polar, sugar-containing lipids that are major constituents of neuronal membranes. Gangliosides are neuroprotective in animal models of neurotoxicity and may also be useful in patients with clinical conditions such as spinal cord injury. We show that a series of gangliosides inhibit nitric oxide synthase activity by binding calmodulin. The prevention of glutamate neurotoxicity in cortical cultures by gangliosides closely parallels their potencies in binding calmodulin and inhibiting nitric oxide synthase. Neuroprotective effects of gangliosides may arise from blockade of nitric oxide formation.     

Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury

Microglial activation has been reported to promote neurotoxicity and also neuroprotective effects. A possible contributor to this dichotomy of responses may be the degree to which proximal neurons are injured. The aim of this study was to determine whether varying the severity of neuronal injury influenced whether microglia were neuroprotective or neurotoxic. We exposed cortical neuronal cultures to varying degrees of hypoxia thereby generating mild (<20% death, 30 min hypoxia), moderate (40-60% death, 2 h hypoxia), or severe (>70% death, 6 h hypoxia) injuries. Twenty-four hours after hypoxia, the media from the neuronal cultures was collected and incubated with primary microglial cultures for 24 h. Results showed that the classic microglial proinflammatory mediators including inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin-1-beta were upregulated only in response to mild neuronal injuries, while the trophic microglial effectors brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were upregulated in response to all degrees of neuronal injury. Microglia stimulated with media from damaged neurons were co-cultured with hypoxic neurons. Microglia stimulated by moderate, but not mild or severe damage were neuroprotective in these co-cultures. We also showed that the severity-dependent phenomenon was not related to autocrine microglial signaling and was dependent on the neurotransmitters released by neurons after injury, namely glutamate and adenosine 5'-triphosphate. Together our results show that severity of neuronal injury is an important factor in determining microglial release of "toxic" versus "protective" effectors and the resulting neurotoxicity versus neuroprotection.