Alterations in basement membrane immunoreactivity of the diabetic retina in three diabetic mouse models.

BACKGROUND: The mouse retina contains three kinds of basement membrane (BM) structures; the inner limiting membrane (ILM), Bruch's membrane (BrM), and the BM surrounding the capillaries. We aimed to investigate possible variations of individual BM components and to detect effects caused by diabetes in three different diabetic mouse models. METHODS: After 4 and 6 months of diabetes (defined by blood glucose > 250 mg/dl), we analyzed by immunohistochemistry the laminin, collagen IV, and nidogen-1 and nidogen-2 protein composition of the BMs obtained from diabetic and non-diabetic Leptin-receptor deficient (db/db) mice and insulin receptor (IR)/insulin receptor substrate-1 (IRS-1) double heterozygous knockout mice. In addition, C57BL/6 J mice were rendered diabetic by intraperitoneal injections of streptozotocin (STZ). RESULTS: All analyzed BM proteins were detected in all of the three BMs with the exception of collagen IV, which was not detectable in the ILM of db/db mice and IR/IRS-1 mice. We present the first analysis of nidogen expression in diabetic BM. The staining patterns did not differ between the type-1 diabetic model (STZ) or the type-2 diabetic models (db/db and IR/IRS-1) and the wild-type controls, with only one exception: both the db/db mice and the IR/IRS-1 mice but not the STZ mice showed a decreased nidogen-1 immunoreactivity in the BrM after 4 months of diabetes, but not after 6 months. CONCLUSIONS: The BMs in the three mouse strains differ with regard to protein immunoreactivity in the inner limiting membrane. Changes in BM composition may affect both the assembly and the function of the retinal BM. However, there are no marked differences in the BM composition between type-1 and type-2 diabetes. These results provide evidence for BM remodelling during diabetic retinopathy.

Iris pigment epithelial cells: a possible cell source for the future treatment of neurodegenerative diseases.

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

Neural precursor cells as carriers for a gene therapeutical approach in tumor therapy.

Conventional therapeutical approaches such as surgery, radiotherapy, or chemotherapy have been shown to be rather unsuccessful in the treatment of infiltrative growing tumors such as the malignant glioblastoma multiforme. Thus, new therapeutical strategies have to be developed that are suitable for inducing cell death also in migrating tumor cells. These new therapeutical stategies include cell and/or gene therapeutical approaches. We demonstrate that glial-restricted progenitor cells as well as embryonic stem cell-derived neural stem cells belong to cell populations applicable to such therapeutical concepts. Both cell types can be efficiently transduced using a third-generation high-capacity "gutless" adenoviral vector, and show a tropism for the F98 glioma cells by migrating towards a spheroid of F98 glioma cells with a tendency to form a barrier around the tumor spheroid in an in vitro tumor confrontation model. Moreover, in a migration assay, secretion products of glial-restricted precursor cells have shown a potency to inhibit the migratory activity of glioma cells in vitro. In vivo, F98 glioma cell-derived tumor formation in the right striatum resulted in migration of glial as well as neural precursor cells towards the tumor area when cotransplanted in the corpus callosum of the contralateral hemisphere. After arrival, both cell types surround the tumor mass and even invade the experimentally induced tumor. These data indicate that glial-restricted as well as embryonic stem cell-derived neural precursor cells are good candidates as carriers for an ex vivo gene therapeutical approach in tumor therapy.

The high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt and 23,000 mol. wt) mediate neurotrophic activity on rat embryonic mesencephalic dopaminergic neurons in vitro.

Basic fibroblast growth factor is expressed in different isoforms which display tissue and species specificity and are differentially regulated during development and after experimental interventions. The differential regulation of the fibroblast growth factor-2 isoforms may indicate specific activities and functions of these molecules. The characterization of fibroblast growth factor-2 effects, however, is almost exclusively based on studies including the 18,000 mol. wt isoform. It is not yet known whether the high molecular weight fibroblast growth factor-2 isoforms (21,000 mol. wt, 23,000 mol. wt) exert similar or distinct activities in the nervous system. In the present study, we investigated the effects of the high molecular weight isoforms on dissociated rat mesencephalic dopaminergic neurons. For this purpose, recombinant fibroblast growth factor-2 isoforms, prepared in a histidine expression system, were administered on dopaminergic neurons in vitro, and Schwann cells over-expressing the high molecular weight isoforms were co-cultured with dopaminergic neurons. This is the first demonstration to show that the high molecular weight isoforms mediate a neurotrophic activity. Exogenous high molecular weight fibroblast growth factor-2 isoforms stimulated the survival of embryonic mesencephalic dopaminergic neurons and protected them from 6-hydroxydopamine neurotoxicity. In addition, co-culture of dopaminergic neurons with high molecular weight fibroblast growth factor-2 over-expressing Schwann cells revealed an increased survival and neurite formation of the mesencephalic dopaminergic neurons. These results suggest that the high molecular weight fibroblast growth factor-2 isoforms may serve as a new tool for the treatment of Parkinson's disease.

S-100beta protects cultured neurons against glutamate- and staurosporine-induced damage and is involved in the antiapoptotic action of the 5 HT(1A)-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.

Neuroprotection mediated via neurotrophic factors and induction of neurotrophic factors.

Neurotrophins and other neurotrophic factors have been shown to support the survival and differentiation of many neuronal populations of the central and peripheral nervous system. Therefore, administering neurotrophic factors could represent an alternative strategy for the treatment of acute and chronic brain disorders. However, the delivery of neurotrophic factors to the brain is one of the largest obstacles in the development of effective therapy for neurodegenerative disorders, because these proteins are not able to cross the blood-brain barrier. The induction of growth factor synthesis in the brain tissue by systemically administered lipophilic drugs, such as beta-adrenoceptor agonists, shown to increase endogenous nerve growth factor (NGF) synthesis in the brain, would be an elegant way to overcome these problems of application. Stimulation of beta-adrenoceptors with clenbuterol led to increased NGF synthesis in cultured central nervous system (CNS) cells and rat brain tissue. Clenbuterol-induced NGF expression was reduced to the control levels by coadministration of beta-adrenoceptor antagonist propranolol. Furthermore, clenbuterol protected rat hippocampal neurons subjected to excitotoxic damage. The neuroprotective effect of clenbuterol in vitro depended on increased NGF synthesis, since the neuroprotection was abolished by NGF antisense oligonucleotide as well as by antibodies directed against NGF itself. In vivo, clenbuterol protected rat hippocampus in a model of transient forebrain ischemia and reduced the infarct volume in a rat model of permanent middle cerebral artery occlusion (MCAo). The neuroprotective effect of clenbuterol in vivo was accompanied by enhanced NGF synthesis in brain tissue. These findings support our hypothesis that orally active NGF inducers may have a potential as therapeutic agents for the treatment of neurodegenerative disorders and stroke.

Ciliary neurotrophic factor enhances the expression of NGF and p75 low-affinity NGF receptor in astrocytes.

A functional interactions between ciliary neurotrophic factor (CNTF) and NGF has recently been demonstrated. We found that the exposure of rat cortical astrocytes to human recombinant CNTF for 3 h increased the level of mRNA for NGF as determined by reverse transcription-polymerase chain reaction (RT-PCR). The increase in NGF message was followed by corresponding increase in NGF protein secreted from the astrocytes into the culture medium as determined 6 h later. C-fos seemed to be involved in the mechanism of NGF induction since the expression of c-fos gene preceded NGF mRNA elevation. Furthermore, we found that in cultured astrocytes exogenous CNTF increased the level of mRNA coding for p75(NTR), the low affinity receptor for NGF and other neurotrophins. CNTF is highly expressed in the lesioned brain and CNTF-induced upregulation of NGF synthesis could be involved in the endogenous repair mechanisms.

Ciliary neurotrophic factor protects hippocampal neurons from excitotoxic damage.

The loss of neurons is responsible for many acute neurological disorders as well as chronic neurodegenerative diseases. This cell loss might be prevented by a direct delivery of neurotrophic factors. Therefore, we investigated the capacity of ciliary neurotrophic factor (CNTF) and nerve growth factor (NGF) as well as the combination of both growth factors on the glutamate-induced excitotoxic damage in hippocampal cultures. The exposure of hippocampal neuronal/glial co-cultures to 0.5 mM L-glutamate for 1 h induced pronounced neurotoxicity evaluated 18 h later by trypan blue staining and morphological criteria. The damaged neurons showed both apoptotic and necrotic features. However, CNTF (1-1000 pg/ml) reduced neuronal degeneration when administered 6 and 24 h before induction of injury and remained in contact with the cells until evaluation of neuronal damage. Furthermore, NGF (1 ng/ml) also rescued the hippocampal neurons under the same experimental conditions and with a similar to CNTF potency. However, the co-administration of NGF and CNTF (but not either factor alone) restored the neuronal survival to control levels. Our results support the hypothesis that administering neurotrophic factors could represent an alternative strategy for the treatment of acute and chronic brain disorders.

NGF mediates the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study.

Previous studies in our laboratory suggested that neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1-100 microM) an increase in nerve growth factor mRNA and protein levels (200-300% of control) was observed in primary cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3-1 microM for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 microM, 30 h), clenbuterol (10 microM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 microM) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. In vivo, clenbuterol (0.01-0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve growth factor antisense oligonucleotides injected into the cortical tissue before ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo.

The 5-HT1A receptor agonist Bay x 3702 inhibits apoptosis induced by serum deprivation in cultured neurons.

We examined whether the highly selective 5-HT1A receptor agonist (-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]-amino]butyl]-11 ,2-benz-isothiazol-3(2H)-one 1,1-dioxide monohydrochloride (Bay x 3702) could inhibit neuronal apoptosis induced by serum deprivation. In primary cultures of chick embryonic neurons and in mixed neuronal/glial cultures from neonatal rat hippocampus, Bay x 3702 (1 microM) rescued serum-deprived neurons from apoptosis. The antiapoptotic effect of Bay x 3702 (1 microM) was blocked in chick neurons by the selective 5-HT1A receptor antagonists 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazin]ethyl]-N-2-pyridinyl-be nzamide hydrochloride (p-MPPI, 10 microM) and 4-[3-benzotriazol-1-propyl]-1-(2-methoxyphenyl)-piperazine (BPMP, 10 microM) as well as by anti-nerve growth factor (anti-NGF) antibodies and in rat neurons by N-[2-4-(2-methoxy)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane-carbo xamide trihydrochloride (WAY 100635, 10 microM). We found only under control conditions (medium with serum), but not in serum-deprived cultures, that NGF secretion was 6-fold increased by Bay x 3702 (1 microM) compared to untreated cultures. Additionally, Bay x 3702 (4 microg/kg i.v.), infused within a period of 4 h, significantly increased the NGF content of the rat hippocampus, but not of the striatum. In summary, our data suggest that Bay x 3702 inhibited growth factor withdrawal-induced apoptosis by the stimulation of 5-HT1A receptors and that the NGF signalling pathway is involved in the mechanism of action.

Neuroprotective effect of 5-HT1A receptor agonist, Bay X 3702, demonstrated in vitro and in vivo.

It has been shown recently that Bay X 3702 ((-)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]amino]butyl]-1, 2,-benzisothiazol-3(2H)-one 1,1-dioxide monohydrochloride), a highly potent and selective 5-HT1A receptor agonist, has a neuroprotective potency associated with its ability to inhibit ischemia-induced excessive release of glutamate. 5-HT1A receptors are highly expressed in brain areas, such as the hippocampus and the cerebral cortex, sensitive to neuronal damage induced by ischemic stroke or brain trauma. Therefore, we investigated whether Bay X 3702 can rescue cultured hippocampal neurons subjected to excitotoxic damage. The hippocampal neurons exposed to 0.5 mM L-glutamate for 1 h had pronounced damage characteristic of neuronal necrosis as evaluated 18 h later by trypan blue staining and morphological criteria. However, treatment with Bay X 3702 (0.001 to 1 microM) reduced the number of damaged neurons, and preserved cell morphology and integrity of the neuronal network. Bay X 3702 was added immediately after the end of exposure to glutamate and was present until the evaluation of neuronal damage. Furthermore, the neuroprotective activity of Bay X 3702 (0.1 microM) was abolished by WAY 100635 (N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl cyclo-hexanecarboxamide) (1 microM), a selective 5-HT1A receptor antagonist, indicating that the neurorescuing activity of Bay X 3702 was mediated via stimulation of 5-HT1A receptors. Additionally, we attempted to find whether the drug could protect rat brain tissue from ischemic insult due to permanent occlusion of the middle cerebral artery in rats. Bay X 3702 (12 and 40 microg/kg), infused within a period of 4 h, immediately after induction of ischemia greatly reduced cortical infarct volume (57 and 55% of controls, respectively) suggesting that this drug might be useful for the treatment of acute cerebral infarction.

Stimulation of beta2-adrenoceptors inhibits apoptosis in rat brain after transient forebrain ischemia.

We have previously demonstrated that the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo was most likely mediated by an increased nerve growth factor (NGF) expression. In the present study, we examined whether clenbuterol was capable of inhibiting apoptosis caused by ischemia. Transient forebrain ischemia was performed in male Wistar rats (300 to 350 g) by clamping both common carotid arteries and reducing the blood pressure to 40 mm Hg for 10 minutes. Clenbuterol (0.1, 0.5, and 1.0 mg/kg intraperitoneally) was administered 3 hours before ischemia or immediately after ischemia. The brains were removed for histologic evaluation 7 days after ischemia. The time course of DNA fragmentation was determined 1, 2, 3 and 4 days after ischemia. Staining with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) was used for further analysis of DNA fragments in situ 3 days after ischemia. The NGF protein was assayed by enzyme-linked immunosorbent assay. Ten-minute forebrain ischemia damaged 80% to 90% of the neurons in the hippocampal CA1 region evaluated 7 days after ischemia. Pretreatment with clenbuterol (0.5 and 1.0 mg/kg) reduced the neuronal damage by 18.1% (P < 0.01) and 13.1% (P < 0.05), respectively. The neuroprotective effect also was found when clenbuterol (0.5 mg/kg) was administered immediately after ischemia (P < 0.05). The DNA laddering appeared in striatum 1 day and in hippocampus 2 days after ischemia and peaked on the third day in both regions. The DNA laddering was nearly abolished in the hippocampus and partially blocked in striatum and cortex by 0.5 mg/kg clenbuterol. These results were confirmed by TUNEL staining. Clenbuterol (0.5 mg/kg intraperitoneally) elevated the NGF protein level by 33% (P < 0.05) in the hippocampus and 41% (P < 0.05) in the cortex 6 hours after ischemia. Three days after ischemia, the NGF levels in these regions were no longer different between the clenbuterol-treated and control groups. This study clearly demonstrates that clenbuterol possesses a neuroprotective activity and a marked capacity to inhibit DNA degradation after global ischemia. The results suggest that clenbuterol increases NGF expression during the first hours after global ischemia and thereby protects neurons against apoptotic damage.

Lubeluzole protects hippocampal neurons from excitotoxicity in vitro and reduces brain damage caused by ischemia.

Previously reported effects of lubeluzole, such as inhibition of glutamate release, inhibition of nitric oxide (NO) synthesis and blockage of voltage-gated Na+- and Ca2+-ion channels, suggest a neuroprotective action of this drug. Here we report about the effects of lubeluzole and its R-isomer on glutamate-induced neuronal cell death in mixed hippocampal cultures. In addition, we studied the effect of lubeluzole in focal cerebral ischemia models in mice and rats. In hippocampal cultures exposed to 500 nM glutamate for 1 h, lubeluzole (0.1-100 nM), but not the R-isomer (1-100 nM), reduced the percentage of damaged neurons from 42 +/- 8% to 18 +/- 7% (P < 0.01). In mice and rats, lubeluzole reduced ischemic brain damage, when administered immediately after middle cerebral artery occlusion. Interestingly, the protective effect (reduction of the infarct volume in rats to 77% of control; P < 0.01) was also found when the lubeluzole treatment (2.5 mg/kg) was started 3 h after ischemia. Especially this latter effect suggests that lubeluzole will be a useful drug for stroke therapy.

Selegiline enhances NGF synthesis and protects central nervous system neurons from excitotoxic and ischemic damage.

It has been previously demonstrated that selegiline, an irreversible monoamine oxidase B (MAO-B) inhibitor, potentiates glial reaction to injury and possesses some 'trophic-like' activities which do not depend on the inhibition of MAO-B and which are probably associated with the induction of astrocyte-derived neurotrophic substances. Based on these findings, we tried to find out whether selegiline is able to modify the expression of nerve growth factor (NGF) and to protect central nervous system (CNS) neurons from excitotoxic and ischemic damage. Selegiline (10 pM-1 nM) induced NGF messenger RNA (mRNA) expression in cultured rat cortical astrocytes as determined by reverse transcription-polymerase chain reaction (RT-PCR) followed by a corresponding increase in NGF protein content measured by two-site NGF-enzyme-linked immunosorbent assay (ELISA) in astrocyte-conditioned medium. Additionally, exposure of hippocampal cultures containing neuronal and glial cells to this drug at the same concentrations enhanced significantly the content of NGF measured in the culture medium after 6 h of incubation. We hypothesize that selegiline could rescue hippocampal neurons from injury by induction of astrocyte-derived NGF in this cell culture system. To test this hypothesis, an excitotoxic damage was induced in the same type of cells by exposure to 0.5 mM L-glutamate for 1 h. Selegiline (10 pM-1 nM) present in the growth medium 6 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement) protected hippocampal neurons from excitotoxic death. Furthermore, administered intraperitoneally (i.p.) (8 x 15 mg/kg per day) this drug enhanced the expression of NGF message in intact rat cerebral cortex and protected rat cortical tissue from ischemic insult due to permanent occlusion of the middle cerebral artery (MCA). The neuroprotective activity of selegiline (5 x 10 mg/kg per day i.p.) was also demonstrated in a mouse model of focal cerebral ischemia. The present data show that selegiline induced NGF expression in cultured rat cortical astrocytes. In mixed primary cultures of hippocampal neuronal and glial cells, selegiline increased NGF protein content and protected hippocampal neurons from excitotoxic degeneration. In vivo, this drug induced NGF gene expression in cerebral cortex from intact rats and protected rat and mouse cortical tissue from ischemic insult after occlusion of the MCA. Our results indicate that the induction of astrocyte-derived NGF could contribute to the neuroprotective activity of selegiline demonstrated both in vivo and in vitro and can explain, in part, the 'trophic-like' properties of this compound which has been observed by others.

Clenbuterol protects mouse cerebral cortex and rat hippocampus from ischemic damage and attenuates glutamate neurotoxicity in cultured hippocampal neurons by induction of NGF.

It has been shown previously that clenbuterol, a beta 2-adrenergic receptor agonist, enhances NGF synthesis in adult rat brain. Since NGF is able to protect neurons against damage, we tried to find out whether clenbuterol can rescue cultured hippocampal neurons from excitotoxic damage by induction of NGF. The neuroprotective activity of clenbuterol on neurons in the vulnerable CA1 subfield of the hippocampus was tested in a rat model of transient forebrain ischemia. Additionally, in the mouse model of focal cerebral ischemia the ability of clenbuterol to reduce the infarct size was examined. Exposure of mixed neuronal/glial hippocampal cultures to clenbuterol (1 to 100 microM) enhanced significantly the content of NGF measured in the culture medium by two-site ELISA. The excitotoxic injury was induced in the same type of cells after 14 days in vitro by exposure to 1 mM L-glutamate for 1 h in serum-free medium. NGF itself (0.15 to 100 ng/ml) added to the growth medium 4 h before until 18 h after induction of injury (the point of glutamate-toxicity measurement), protected hippocampal neurons from excitotoxic damage. Clenbuterol (1 to 100 microM) provided similar neuroprotection as NGF under the same experimental conditions. The neuroprotective activity of clenbuterol (100 microM) against glutamate-induced damage in hippocampal cultures was blocked by anti-NGF monoclonal antibodies (0.5 microgram/ml) added to the medium during the clenbuterol exposure, demonstrating that the neuronal rescue is mediated by NGF. Propranolol, a beta-adrenergic receptor antagonist (10 microM) added 20 min before and kept in the medium during exposure of the cultures to clenbuterol (1 microM) reversed the neuroprotective activity, suggesting that the induction of NGF and neuroprotection caused by clenbuterol are mediated via beta-adrenergic receptor activation. The capacity of clenbuterol to protect hippocampal neurons was also demonstrated in vivo in a rat model of transient forebrain ischemia. Clenbuterol (4 x 1 mg/kg) administered intraperitoneally increased the number of viable neurons in CA1 subfield of the rat hippocampus. Furthermore, clenbuterol (0.3 and 1 mg/kg, i.p. and 1 mg/kg, s.c.) reduced significantly the infarct area on the mouse brain surface after occlusion of the middle cerebral artery. The present data demonstrate that clenbuterol induces NGF synthesis in cultured hippocampal cells and protects hippocampal neurons from excitotoxic damage. The neuroprotective activity of clenbuterol is also demonstrated in vivo in two rodent models of cerebral ischemia. The results offer strong evidence that the neuroprotective activity of clenbuterol is caused by activation of beta-adrenergic receptors and the subsequent increased expression of NGF.

Central effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) in mice.

The central effects of endothelin-1 (ET-1, 0.5, 1, 2.5 and 10 pmol/mouse) and endothelin-3 (ET-3, 2.5, 5, 10, 20 and 25 pmol/mouse) have been studied after intracerebroventricular (i.c.v.) administration in mice. The following methods were used: behavioral observations, rotarod, spontaneous and amphetamine-stimulated motor activity, and hexobarbital-induced narcosis. ET-1 and ET-3 evoked a dose-dependent behavioral syndrome with a short excitatory period in the higher doses lasting for 5-10 min, followed by a prolonged depressive phase (about 90 min). ET-1 and ET-3 produced central depressive effects demonstrated by depressive behavior signs, decrease of the spontaneous and amphetamine-stimulated motor activity, and prolongation of the hexobarbital-induced narcosis.

L-NAME augments the antinociceptive effects of intracerebroventricularly applied ET-1 and ET-3.

The interaction between endothelin-1 (ET-1) (5 pmol/mouse, i.c.v.) and endothelin-3 (ET-3) (5 pmol/mouse, i.c.v.) with NG-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg i.p., 30 min pretreatment) was investigated in mice by the use of two experimental procedures: hot plate and tail flick tests in mice. L-NAME showed slight insignificant antinociceptive action, but augmented significantly the antinociceptive effects of i.c.v. administered ET-1 and ET-3 in both experimental tests.

Effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on convulsive seizures.

Endothelins (ET-1 and ET-3) are produced in the brain and may act as neuropeptides. The effects of two endothelins (ET-1 and ET-3) were investigated using pentylenetetrazole- (PTZ), strychnine- (STR), picrotoxin- (PIC) and bicuculline-induced (BIC) convulsions. ET-1 did not exert any anticonvulsive effect. ET-3, being more specific for the brain, showed anticonvulsive activity on PTZ and BIC seizures, but not on PIC and STR seizures. Different mechanisms underlie the action of the convulsants. PTZ, BIC and PIC realized their convulsive effects through the GABAergic inhibitory transmitter system; STR exerted an excitatory effect on the spinal cord by blocking the glycine transmitter system. The results suggest that the anticonvulsive effects of ET-3 might be due to an indirect influence on the GABAergic system in the brain.

Antinociceptive effect of centrally administered endothelin-1 and endothelin-3 in the mouse.

The antinociceptive effects of centrally administered (i.c.v.) endothelin-1 (ET-1) and endothelin-3 (ET-3) were studied in mice by the use of 3 experimental procedures: hot plate, tail flick and acetic acid writhing tests. ET-1 (0.625-5 pmol/mouse) and ET-3 (2.5-25 pmol/mouse) produced statistically significant increase of the hot plate and tail flick latencies with duration of about 120 min. ET-3 showed weaker antinociceptive effect. ET-1 inhibited acetic acid-induced writhings with ED50 = 1.9 (1.1-2.7) pmol/mouse. With ET-3 a maximum effect of 45.2% suppression of the writhing response was achieved at 5 pmol/mouse. The antinociception due to ET-1 and ET-3 was not antagonized by naloxone and is thus independent of endogenous opioid release.

Studies on the effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) in brain hypoxia and on the participation of brain prostanoids in their actions.

The effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) in brain hypoxia have been studied in mice using the following experimental models: hypobaric hypoxia induced by low atmospheric pressure, histotoxic hypoxia induced by 12.5 mg/kg KCN i.p., and complete ischemia induced by decapitation. ET-1 and ET-3 were injected intracerebroventricularly (i.c.v.) 15 min before the tests. Forebrain tissue concentrations of 6-keto-PGF1 alpha and thromboxane B2 (TxB2) were measured 15 min following i.c.v. administration of ET-1 (5 pmol/mouse) and ET-3 (10 pmol/mouse). ET-1 (1-5 pmol/mouse) and ET-3 (5-25 pmol/mouse) showed a dose-dependent increase in the survival/gasping time in all models of hypoxia. The effect reached its maximum between 15 and 30 min after ET administration and lasted for about 120 min. ET-1 and ET-3 did not significantly change the brain levels of 6-keto-PGF1 alpha and TxB2. The protective effect of ET-1 and ET-3 was unexpected, because endothelins (ETs) are the most potent vasoconstrictors known, and in doses close to those used in this study they cause vasoconstriction and decrease in cerebral blood flow. The protection was not likely to be due either to stimulation of the endogenous release of prostacyclin (PGI2) or to a decrease in the deleterious prostanoid thromboxane A2 (TxA2). Additional experiments are necessary to explain the cerebroprotective effects of ET-1 and ET-3.