Josef Krieglstein

In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.

A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.

Transforming Growth Factor-β1 Increases Bad Phosphorylation and Protects Neurons Against Damage

Despite the characterization of neuroprotection by transforming growth factor-β1 (TGF-β1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-β1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-β1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser112 in mouse brains after adenovirus-mediated TGF-β1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-β1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-β1 inhibited the increase in Bad expression caused by staurosporine. TGF-β1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-β1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-β1 in cultured hippocampal cells. Together, we demonstrate that TGF-β1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

Transforming Growth Factor-β1 Prevents Glutamate Neurotoxicity in Rat Neocortical Cultures and Protects Mouse Neocortex from Ischemic Injury in vivo

Transforming growth factor-β1 (TGF-β1) has been shown to be an injury-related peptide growth factor within the mammalian central nervous system. We tested whether TGF-β1 has the capacity to protect rat neocortical neurons against excitotoxic damage in vitro and mouse neocortex against ischemic injury in vivo. After 14 days in vitro, cultured neurons from rat cerebral cortex were exposed to 1 mM l-glutamate in serum-free culture medium. The cultures received TGF-β1 immediately after the addition of glutamate. Eighteen hours later, the cell viability of the cultures was determined using trypan blue exclusion. TGF-β1 (1–10 ng/ml) significantly reduced the excitotoxic neuronal damage in a concentration-dependent manner. In vivo, male NMRI mice were subjected to a permanent occlusion of the left middle cerebral artery by microbipolar electrocoagulation. After 48 h, the animals received a transcardiac injection of carbon black. The area of ischemia (devoid of carbon) was restricted to the neocortex and its size was determined planimetrically by means of an image-analyzing system. The treatment with TGF-β1 (1 μg/kg i.c.v.) at 6, 4, or 2 h prior to vessel occlusion reduced the area of ischemia by 5.3, 10.0, and 9.6%, respectively. The effect of the treatment with TGF-β1 was statistically significant (p < 0.05 by two-way ANOVA). The present in vitro and in vivo data suggest that TGF-β1 has the capacity to diminish the deleterious consequences of an excitotoxic or ischemic insult.

TGF-β1 Protects Hippocampal Neurons Against Degeneration Caused by Transient Global Ischemia Dose-Response Relationship and Potential Neuroprotective Mechanisms

BACKGROUND AND PURPOSE Transforming growth factor-beta 1 (TGF-beta 1) has been shown to rescue cultured neurons from excitotoxic and hypoxic cell death and to reduce infarct size after focal cerebral ischemia in mice and rabbits. The present study investigated the effects of TGF-beta 1 in a different pathophysiological setting and the delayed neuronal death of hippocampal pyramidal cells after transient global ischemia in rats, and evaluated the potential mechanisms of the neuroprotective activity of TGF-beta 1. METHODS Transient forebrain ischemia was induced in male adult Wistar rats with bilateral occlusion of both common carotid arteries combined with systemic hypotension for 10 minutes. Seven days after ischemia, brains were perfusion-fixed and stained for histological evaluation. TGF-beta 1 or vehicle was injected intracerebroventricularly (ICV; 0.5, 4, and 50 ng) or intrahippocampally (4 ng) 1 hour before ischemia. For in vitro studies, hippocampal neurons were derived from E17 rat embryos and cultured for 10 to 14 days. Cells were exposed to (1) S-nitrosocysteine (SNOC; 30 mumol/L) to induce nitric oxide-induced oxidative injury and (2) staurosporine (0.03 mumol/L) to induce apoptotic cell death. RESULTS Transient forebrain ischemia caused extensive degeneration of CA1 hippocampal pyramidal cells in vehicle-treated control animals. Ischemic injury was not significantly reduced after ICV administration of 0.5 ng TGF-beta 1 (71 +/- 7% damaged neurons versus 84 +/- 3% in vehicle-treated controls: n = 9 and 11, respectively; P = .07, Mann-Whitney U test). Administration of 4 ng TGF-beta 1 reduced the percentage of damaged CA1 pyramidal cells from 71 +/- 10% in controls to 52 +/- 7% in TGF-beta 1-treated animals (n = 11 and 12, respectively; P = .04). TGF-beta 1 (4 ng) also produced significant protection when injected directly into the hippocampal tissue. In contrast, ICV administration of 50 ng TGF-beta 1 failed to show a protective effect in two separate sets of experiments. In vitro, a 24-hour pretreatment of the cultured hippocampal neurons with TGF-beta 1 (0.1 to 10 ng/mL) significantly inhibited both nitric oxide and staurosporine neurotoxicity. Posttreatment with TGF-beta 1 (10 ng/mL) also inhibited staurosporine neurotoxicity but actually potentiated nitric oxide-induced neuronal injury. CONCLUSIONS We demonstrated that TGF-beta 1 in a surprisingly low dose range has the capacity to reduce injury to CA1 hippocampal neurons caused by transient global ischemia in rats. This protective action could well be associated with the antioxidative and antiapoptotic effects of TGF-beta 1 demonstrated in vitro.

Neuroprotection by Estrogens in a Mouse Model of Focal Cerebral Ischemia and in Cultured Neurons: Evidence for a Receptor-Independent Antioxidative Mechanism

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or β-amyloid. Here, the authors report that 17β-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.003 to 30 mg/kg) reduced brain tissue damage after permanent occlusion of the middle cerebral artery in male NMRI mice. In vitro, 17β-estradiol (1 to 10 μmol/L) and 2-OH-estradiol (0.01 to 1 μmol/L) reduced the percentage of damaged chick embryonic neurons treated with FeSO4. In these primary neurons exposed to FeSO4, the authors also found reactive oxygen species to be diminished after treatment with 17β-estradiol (1 to 10 μmol/L) or 2-OH-estradiol (0.01 to 10 μmol/L), suggesting a strong antioxidant activity of the estrogens that were used. Neither the neuroprotective effect nor the free radical scavenging properties of the estrogens were influenced by the estrogen receptor antagonist tamoxifen. The authors conclude that estrogens protect neurons against damage by radical scavenging rather than through estrogen receptor activation.

S-100β protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT1A-receptor agonist, Bay x 3702

The serotonin (5-HT)(1A) receptor agonists have already been shown to protect cultured neurons from excitotoxic as well as from apoptotic damage [B. Ahlemeyer, J. Krieglstein, Stimulation of 5-HT(1A) receptors inhibits apoptosis induced by serum deprivation in cultured neurons from chick embryo, Brain Res. 777 (1997) 179-186. ; B. Ahlemeyer, A. Glaser, C. Schaper, I. Semkova, J. Krieglstein, The 5-HT(1A) receptor agonist, Bay x 3702, inhibited apoptosis induced by serum deprivation in cultured neurons, Eur. J. Pharmacol. 370 (1999) 211-216.; J.H.M. Prehn, M. Welsch, C. Backhauss, J. Nuglisch, F. Ausmeier, C. Karkoutly, J. Krieglstein, Effects of serotonergic drugs in experimental brain ischemia: evidence for a protective role of serotonin in cerebral ischemia, Brain Res. 630 (1993) 110-120.; I. Semkova, P. Wolz, J. Krieglstein, Neuroprotective effect of 5-HT(1A) receptor agonist, Bay x 3702, demonstrated in vitro and in vivo, Eur. J. Pharmacol. 359 (1998) 251-260.; B. Suchanek, H. Struppeck, T. Fahrig, The 5-HT(1A) receptor agonist, Bay x 3702, prevents staurosporine-induced apoptosis, Eur. J. Pharmacol. 355 (1998) 95-101.] and to increase the release of the neurotrophic protein, S-100beta [P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, Stimulation of astroglial 5-HT(1A) receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology, Brain Res. 497 (1989) 80-86. ; P.M. Whitaker-Azmitia, R. Murphy, E.C. Azmitia, S-100 protein is released from astroglial cells by stimulation of 5-HT(1A) receptors, Brain Res. 528 (1990) 155-158.]. In this study, we tried to find out whether S-100beta can protect cultured neurons from glutamate- and staurosporine-induced damage and whether the neuroprotective activity of the highly selective 5-HT(1A) receptor agonist, Bay x 3702, is mediated by an induction of S-100beta. Extracellularly added S-100beta (1-10 ng/ml) reduced staurosporine-induced damage in pure neuronal cultures from chick embryo telencephalon as well as in mixed neuronal/glial cultures from neonatal rat hippocampus. In addition, S-100beta (1 ng/ml) reduced neuronal death induced by exposure to glutamate (0.25 mM, 30 min) in mixed neuronal/glial cultures from neonatal rat hippocampus. In cultured rat cortical astrocytes, a 24 h-treatment with Bay x 3702 (1 nM) increased the S-100beta content in the culture medium from 2.2+/-0.3 (controls) to 6.2+/-0.7 ng/ml. In the adult rat, a 4 h-infusion of 4 microg/kg Bay x 3702 (i.v.) was found to increase the S-100beta content in the striatum 6 h after the beginning of the infusion to 153+/-37 microg/g compared with 60+/-20 microg/g in vehicle-treated rats. Bay x 3702 had no effect on the S-100beta content in the rat hippocampus. Finally, we tried to block the protective effect of Bay x 3702 against staurosporine-induced damage in mixed neuronal/glial cultures from rat neonatal hippocampus by anti-S-100beta antibodies. We found only a partial blockade, although the antibodies fully blocked the antiapoptotic effect of S-100beta itself demonstrating that the antibody was effective in blocking neuroprotection by S-100beta. Thus, we conclude that S-100beta was able to protect cultured neurons against glutamate- and staurosporine-induced damage. Furthermore, S-100beta mediated partially the protective effect of the 5-HT(1A) receptor agonist, Bay x 3702, against staurosporine-induced apoptosis in mixed neuronal/glial cultures from neonatal rat hippocampus.

Nerve growth factor survival signaling in cultured hippocampal neurons is mediated through TRKA and requires the common neurotrophin receptor P75

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within 10 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-phosphate (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) 1/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of sphingosine-kinase blocked the neuroprotective effect of NGF, suggesting that sphingosine-1-phosphate was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.

Neuroprotection by transforming growth factor-β1 involves activation of nuclear factor-κB through phosphatidylinositol-3-OH kinase/Akt and mitogen-activated protein kinase-extracellular-signal regulated kinase1,2 signaling pathways

Prevention of neuronal apoptosis has been introduced as a new therapeutic strategy for neurodegenerative disorders. We have previously reported anti-apoptotic effects of transforming growth factor-beta1 (TGF-beta1), a multifunctional cytokine, in models of cerebral ischemia and in cultured neurons and recently focused on the mechanisms underlying the anti-apoptotic effect of TGF-beta1. The anti-apoptotic transcriptional factor nuclear factor kappa B (NF-kappaB) shows high impact in the cell survival function of multiple cytokines and growth factors. The present study explored whether NF-kappabeta is a target of TGF-beta1 and which signaling pathways involved in the activation of NF-kappabeta are triggered by TGF-beta1. We demonstrated that TGF-beta1 increased the transcriptional activity of NF-kappabeta in cultured hippocampal neurons in a time- and concentration-dependent manner. Furthermore, TGF-beta1 induced translocation of p65/NF-kappabeta to the nucleus and enhanced NF-kappabeta transcriptional activity in the presence of apoptotic stimuli. TGF-beta1-mediated NF-kappabeta activation was blocked by wortmannin and U0126, indicating the involvement of both phosphatidylinositol-3-OH kinase (PI3k)/Akt and mitogen-activated protein kinase (MAPK)/extracellular-signal regulated kinase (Erk)1,2 pathways in the action of TGF-beta1. TGF-beta1 produced a concomitant increase in the phosphorylations of Ikappabeta kinase (IKKalpha/beta) and Ikappabetaalpha with a subsequent degradation of Ikappabetaalpha. Interestingly, the increased phosphorylation of IKKalpha/beta and Ikappabetaalpha was abrogated by wortmannin, but not by U0126, suggesting that PI3k/Akt and MAPK/Erk1,2 pathways triggered by TGF-beta1 regulated the activation of NF-kappabeta through different mechanisms. Of note, wortmannin and U0126, as well as kappabeta-decoy DNA, abolished the anti-apoptotic effect of TGF-beta1, corroborating the notion that both PI3k/Akt and MAPK/Erk1,2 pathways, and NF-kappabeta activity are necessary for the anti-apoptotic activity of TGF-beta1.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-κB

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor‐κB (NF‐κB) and how this activation contributes to preconditioning‐induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine‐induced (200 nm, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF‐κB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IκBα as well as by electrophoretic mobility shift assay. Preconditioning‐mediated neuroprotection was abolished by antioxidants, inhibitors of NF‐κB activation and cycloheximide suggesting the involvement of ROS, an activation of NF‐κB and de novo protein synthesis in preconditioning‐mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn‐superoxide dismutase which could be blocked by antioxidants, cycloheximide and κB decoy DNA. Our data suggest that inhibition of staurosporine‐induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF‐κB and an increase in the protein level of Mn‐superoxide dismutase.

Neuroprotective effect of memantine demonstrated in vivo and in vitro

The purpose of the present study was to test whether the anticonvulsant, memantine (1-amino-3,5-dimethyladamantane), can protect neurons against hypoxic or ischemic damage. To this end, we used a rat model of transient forebrain ischemia and cultured neurons from chick embryo cerebral hemispheres. Ischemia was induced for 10 min by clamping both carotid arteries and lowering the mean arterial blood pressure to 40 mm Hg; the rats were allowed to recover for 7 days. Cultured neurons were made hypoxic with 1 mmol/l NaCN added to the incubation medium for 30 min followed by a recovery period of 3 days. The possible effects of memantine were compared with those produced by a typical non-competitive NMDA antagonist, dizocilpine. Similar effects were obtained with both drugs. The drugs reduced the damage caused by transient ischemia to neurons of the hippocampal CA1 subfield. Memantine (10 and 20 mg/kg) had a dose-dependent effect when administered intraperitoneally to the rats 1 h before ischemia. Dizocilpine was active in this model at a dosage of 1 mg/kg. When administered after ischemia, 10 mg/kg memantine significantly protected CA1 neurons against ischemic damage. Furthermore, the drugs protected cultured neurons against hypoxic damage. The lowest effective concentration was 0.1 mumol/l for dizocilpine and 1 mumol/l for memantine. Thus, memantine possesses neuroprotective activity but is less potent than dizocilpine.