bluuebunny/crossref_metadata_2025 huggingface dataset:
Can the developing striatum effect the dopaminergic features of hNT neurons in vitro?
Authors: Misiuta I. , McGrogan M. , Willing A. , Sanberg P. , Zigova T.
Published: May 2003

Our laboratory is investigating the dopaminergic (DA) characteristics of the human, neuronally committed hNT cells for the DA replenishment therapies, like Parkinson's disease. Recent in vitro experiments indicated that hNT cells express markers typical of DA neurons in the substania nigra (SN) including tyrosine hydroxylase (TH), aldehyde dehydrogenase and transcription factor Nurr1. Even though up to 50% of cultured hNT neurons express TH, the long‐term maintenance of DA phenotype may depend on additional factors. Since the striatum is a target tissue for DA neurons in the SN, the goal of the present study is to examine the effect of the developing striatum on the TH expression of hNT neurons in a simplified coculture system.

Acknowledgement: Supported by the Shimberg Scholar Award.
Structural investigations of nicotinic acetylcholine receptor and its intracellular domain
Authors: Kukhtina V. , Brosig A. , Bandini G. , Welte W. , Hucho F.
Published: May 2003

In order to study the conformation of the nicotinic acetylcholine receptor (nAChR), we follow two routes: (a) crystallization of the intact nAChR isolated from Torpedo electric organ and (b) heterologous expression of the intracellular domain of nAChR. (a) In order to produce nAChR solutions that meet known prerequirements for protein crystallization (a monodisperse solution of mixed nAChR‐detergent‐micelles without contamination by other proteins) we will adapt the purification protocol, focusing on the removal of the associated protein rapsyn and deglycosylation. (b) We found that the Torpedo delta subunit intracellular loop can be expressed in E. coli relatively easily, whereas we obtained no expression of the a7 subunit loop, possibly due to its high cysteine content. Therefore our purification attempts will focus on the delta subunit. The purified loop domain will be analyzed using NMR spectroscopy.
Nicotine indirectly increases acetylcholine release in rat striatum
Authors: Machová E. , Žemličková V. , Doležal V.
Published: May 2003

We investigated the influence of nicotine on the release of ACh from rat striatal slices in the presence of domperidone to remove dopamine inhibition of ACh release. In medium facilitating glutamatergic transmission (with glycine and without Mg) nicotine increased the release of ACh evoked by mild potassium depolarization. This effect of nicotine was not present in resting conditions or when the tissue was fully depolarized. Enhancement of the release of ACh was prevented by tetrodotoxine, nicotinic antagonist mecamylamine, and glutamate NMDA antagonist MK 801. Our results suggest that nicotine increases ACh release indirectly by stimulation of nicotinic receptors on glutamatergic nerve terminals leading to the enhancement of glutamate release. Released glutamate in turn increases firing of cholinergic interneurons and ACh release. Nicotinic receptors on striatal glutamatergic nerve terminals perhaps play a role in the mechanism of striatal cholinergic hyperactivity observed in Parkinson's disease.

Acknowledgements: Supported by grants GACR 305/01/0283 and GAAV 5011206.
Effects of NGF on different phenotypes and genotypes of cholinergic murine SN56 cells
Authors: Bielarczyk H. , Szutowicz A. , Madziar B. , Jankowska A.
Published: May 2003

Nerve growth factor (NGF) is important for differentiation and maintenance of septal cholinergic neurons. It caused concentration‐dependent increase of choline acetyltransferase (ChAT) activity ([EC50%] 1 ng/mL), acetylcholine (ACh) content and morphologic maturation of SN56TrkA(+)p75(+) but not TrkA(–)p75(+) cells. NGF added with cyclic AMP altered significantly differential effects of the latter neither in TrkA(–) nor TrkA(+). However, when cyclic AMP‐predifferentiated cells were treated with NGF alone, it caused suppression of the cholinergic phenotype in both cell lines. Anti‐p75 antibodies totally reversed inhibitory effects of NGF on ChAT activity. Differentiation was accompanied by increase whereas its reversal by decrease of intracellular Ca content. These data indicate that NGF may exert opposite effects on phenotype of cholinergic neurons by p75 receptor signaling pathways and changes in intracellular Ca.

Acknowledgement: Supported by KBN project 6P05A 01020.
Learning‐dependent regulation of beta2 and gamma2 subunits of GABAAR mRNA in sensory cortex of mice
Authors: Lech M. , Skibińska A. , Kossut M.
Published: May 2003

GABAA receptors are heteromeric, ligand‐gated ion channels, built up with 19 different subunits. In the somatosensory cortex the most prevalent subunits are: alpha1, beta2 and gamma2. We showed that classical conditioning, in which stimulation of a row of vibrissae (CS) was paired with a tail shock (UCS), results in changes in cortical inhibitory system. Among others, GABAAalpha1 mRNA is up‐regulated in layer IV of cortical representation of trained row of vibrissae. We examined the mRNA expression level of beta2 and gamma2 subunits of GABAAR. For in situ hybridization, 35S‐labeled oligonucleotides were used as antisense probes. The effects were examined 1 h, 24 h and 5 days after the training lasting 3 days. There are no changes observed in beta2 subunits mRNA level. Expression of mRNA of gamma2 subunits increased 5 days after the training, but in contrast to other elements of gabaergic system we investigated, the change was observed in layers II/III. This indicates that learning‐dependent regulation of GABAA receptor phenotype is specific for given neuronal subtype.
Are NMDA receptors in the cortex affected by sensory learning?
Authors: Skibińska A. , Lech M. , Kossut M.
Published: May 2003

NMDA receptor is a heteromeric, ligand‐gated calcium channel, believed to mediate mechanisms of neuronal plasticity. In the adult cerebral cortex the receptor is build up with NR1, NR2A and NR2B subunits. The properties of the receptor are regulated by changing the relative expression of NR2A and NR2B subunits. We have previously found that 3 days of classical conditioning in which stimulation of a row of vibrissae was paired with a tail shock induces NMDA‐receptor dependent changes in cortical representations of the stimulated vibrissae. Expression level of NR2A and NR2B subunits during training was examined by in situ hybridization. NR2A mRNA expression showed a rising tendency in the barrel (layer IV) of the ‘trained’ row of vibrissa after sensory conditioning. At the same time point, NR2A protein level in P2 fraction showed similar increasing tendency. NR2B expression level did not change. The results indicate that a short lasting learning‐dependent task induces a small modification in the composition of NMDA receptor channel complex.
Group I metabotropic glutamate receptors participate in homocysteine‐evoked neurotoxicity
Authors: Ziemińska E. , Stafiej A. , Łazarewicz J. W.
Published: May 2003

Homocysteine (HCY) neurotoxicity is known to be mediated by disturbances in methylation processes and by the NMDA receptor‐mediated excitotoxicity. In this study we examined the role of group I metabotropic glutamate receptors (mGlyRs) in HCY‐induced toxicity in cultured rat cerebellar granule neurons exhibited for 30 min, or cultured in the presence of HCY for 3 days. Acute neurodegeneration induced by >10 mm D,L‐HCY, or subchronic damage evoked by 75–500 µm HCY were enhanced by 50 µm glycine and moderately inhibited by 0.5 µm MK‐801, revealing the NMDA receptor‐mediated portion of HCY neurotoxicity. Also group I mGluRs antagonists 25 µm LY367385 or MPEP given alone only moderately inhibited acute and subchronic HCY neurotoxicity, but given together with MK‐801 completely prevented neurotoxicity. These results demonstrate that acute homocysteine‐induced neurotoxicity is mediated cooperatively by group I mGluRs and NMDA receptors.
Ultrastructural pattern of motor‐neurone degeneration in model of slow neurotoxicity in vitro
Authors: Matyja E. , Taraszewska A. , Nagańska E. , Rafałowska J.
Published: May 2003

The chronic glutamate neurotoxicity is suggested to be involved in selective loss of motor neurones (MN) in amyotrophic lateral sclerosis (ALS). Evidence indicates that the loss of MN might be in part due to defective glutamate (GLU) transport resulting in elevation of synaptic glutamate. The in vitro model of slow neurodegeneration based on inhibition of GLU transport was used to evaluate the ultrastructural characteristics of neuronal and glial toxicity. The organotypic cultures of rat lumbar spinal cord maintaining the neurone–astrocyte metabolic interactions were subjected to GLU uptake blockers: threohydroxyaspartate (THA) and l‐transpyrrolidine‐2,4‐dicarboxylate (PDC). The MN exhibited vacuolar degeneration accompanied by presence of apoptotic profiles. The astroglial cells revealed some morphological abnormalities indicating their damage. The results suggest the involvement of both neuronal and glial components in the process of GLU‐mediated toxicity.
Effect of loss of the locus coeruleus noradrenergic projections by DSP‐4 treatment on striatal dopamine D2 receptors
Authors: Terasmaa A. , Eller M. , Fuxe K. , Harro J. , Rinken A.
Published: May 2003

DSP4 induced lesions of locus coeruleus (LC) noradrenergic neurons influence the ascending mesencephalic dopaminergic (DA) systems by reducing striatal DA turnover and inducing behavioral supersensitivity to dopaminergic drugs, the latter effect being similar to that observed after the loss of striatal DA terminals. Here a similar increase of the density of striatal DA D2 receptors (D2R) without changes in affinities of [3H]raclopride and DA was found in the case of both lesions. 6‐OHDA induced DA lesions caused increases in DA‐dependent activation of [35S]GTPγS, while DSP‐4 had no effect on this effector system. The results indicate that an up‐regulation of striatal D2R density develops after a loss of the LC noradrenergic system as also is the case after DA denervation of the striatum. In contrast, such NA lesions had no effect on D2R‐G protein coupling as found after DA denervation.
Beta‐amyloid peptide is toxic to cholinoceptive neurones in vivo via indirect mechanisms
Authors: Gonzalez I. , Gonzalo P. , Gonzalo‐Ruiz A.
Published: May 2003

The effects of amyloid‐beta (Aβ) protein on the expression of m1, m2 subunits of mAChR and on α7nAChR were analyzed in the cerebral cortex and in the hippocampus of rats following injections of Aβ (1–40) (BACHEM, 2 μg in 1 μL of PBS) into the left retroesplenial cortex (RSg) and injections of 1 μL of PBS into the right RSg. Sections were immunoreacted for the localization of α7, m1, m2, GABA, somatostatin and parvalbumin. Injections of Aβ resulted in loss of neurones expressing α7‐ and m1‐like immunoreactivity (IR) in frontal, RSg cortices, hippocampus and subicular complex. A decrease of α7, m1‐ and m2‐like‐IR fibers and structures‐like terminals was also seen in hippocampus, subicular and cerebral cortex. α7nAChR and m1, m2 subuntis of mAChRs were most commonly identified on GABAergic interneurones. These results point to an effect of Aβ on the synthesis of α7nAChR and mAChRs and suggest an important role of cholinoceptive interneurones in the dysfunction of hippocampus and cerebral cortex seen in AD.
Activation of matrix metalloproteinase‐2 and ‐9 in immature rat brain after cerebral hypoxia‐ischemia
Authors: Makarewicz D. , Ziemka‐Nałęcz M. , Kobel‐Borysiuk A. , Zalewska T.
Published: May 2003

In experimental models of adult brain injury metalloproteinase‐2 and ‐9 (MMPs) are up‐regulated and may play critical role in the pathophysiology of neuronal degeneration. Accordingly, the aim of this study was to evaluate the temporal pattern of MMPs activation following hypoxia‐ischemia (HI) during the early postnatal weeks, when the brain exhibits elevated levels of plasticity and neuroprotection. Alterations in MMPs were assessed over the course of 2 weeks after the insult. Our results show that HI induced in 7‐day‐old rats leads to a fast increase of MMPs activity in ipsilateral hemisphere, with maximum level within the first 48 h after injury. It was much more pronounced in the case of MMP‐9. After the transient decrease at 72 h after HI, the MMPs activity remained elevated again for up to 14 days. This study suggests an involvement of metalloproteinases in the development of immature brain injury after HI.
Protein aggregation in postsynaptic density after transient brain ischemia
Authors: Beręsewicz M. , Zabłocka B. , Domańska‐Janik K.
Published: May 2003

Brief cerebral ischemia causes changes in synaptic transmission and in consequence in neuronal function manifested in delayed cell death of CA1 hippocampal region. Postsynaptic density (PSD) is composed by a network of interacting proteins, including scaffolding proteins, neurotransmitter receptors, cytoskeletal proteins and protein kinases. PSD dynamically modulates signal transduction what influence the cell fate. We investigated the composition of the PSD network and effect of ischemia on its complexity. Two experimental procedures were applied. The interaction between PSD‐95 and Src, Fyn, Raf‐1, paxilin or NMDA receptor subunits were explored using coimmunoprecipitation method. In addition, the effect of ischemia‐reperfusion on the density of PSD were evaluated by measurement of is solubility. We find out the decrease in solubility of the PSD‐95, NR2A, NR2B and Raf‐1. Of interest, the latter was restricted to surviving regions of hippocampus.

Acknowledgement:  Financed by PBZ‐KBN‐002/CD/P05/2000.
Proteomics in neurodegenerative diseases
Authors: Sagi D. , Zabel C.
Published: May 2003

Many neurodegenerative diseases share a common feature: aggregation and deposition of abnormal proteins. The cellular mechanisms that cause these diseases can be studied by animal models. A novel approach to determine changes in protein expression is proteomics, i.e. separation of complex protein mixtures by two‐dimensional‐electrophoresis and subsequent identification of the proteins by mass spectrometry. In this contribution Huntington's and Parkinson's disease have been studied in two distinct mouse model systems using this approach. We identified disease‐related proteins further upstream or downstream of the main disease pathway and proteins changed as a consequence of disease. Moreover, we found up‐/down‐regulated proteins which both diseases have in common. By comparing these results with protein changes associated with ageing in mice we identified shared protein changes as well. These data indicate that neurodegenerative diseases and ageing possibly affect the same cellular pathways.
Mechanisms of dantrolene‐induced neuroprotection against excitotoxic injury
Authors: Makarewicz D. , Ziemińska E. , Łazarewicz J. W.
Published: May 2003

Dantrolene – an inhibitor of ryanodine receptors and calcium stabilizer – prevents ischemia‐ and excitotoxicity‐evoked neurodegeneration. To elucidate the mechanisms of this phenomenon, we investigated effects of dantrolene on the NMDA‐ and glutamate‐induced lesion and stimulation of 45Ca uptake in primary cultures of rat cerebellar granule neurons. Neurodegeneration was evaluated after 24 h with the propidium iodide staining. Bcl‐2 immunoreactivity in cell homogenates was measured by immunoblotting. The results demonstrated that dantrolene applied at micromolar concentrations inhibits in a dose‐dependent manner NMDA‐ and glutamate‐evoked 45Ca uptake in neurones and induces neuroprotection. This effect was additive to known effects of DMSO, a vehicle to dantrolene. Dantrolene failed to induce changes in Bcl‐2 immunoreactivity. Thus, dantrolene‐induced neuroprotection against excitotoxicity may be at least partially mediated by its inhibitory effect on the NMDA receptors.
Tumor necrosis factor‐alpha aggravates hippocampal damage in rat induced by amyloid beta‐peptide (25–35)
Authors: Stepanichev M. , Yakovlev A. , Onufriev M. , Lazareva N. , Zarubenko I. , Gulyaeva N.
Published: May 2003

Male Wistar rats received intrahippocampal injections of 3 nmol aggregated Aβ (25–35), intracerebroventricular injections of 0.5 μg human recombinant TNFα or both. Aβ or Aβ + TNFα (but not TNFα alone) induced cell loss in the CA1 pyramidal cell layer, the extension of neuronal degeneration being significantly larger in Aβ + TNFα‐treated group. In hippocampi of both Aβ‐treated groups (but not in TNFα alone‐treated group) reactive gliosis and microglia activation (upregulation of GFAP and isolectin B4 expression) were demonstrated. TUNEL‐positive nuclei were found in the hippocampi of both Aβ‐treated groups. Caspase‐3 activity significantly increased in the ipsilateral hippocampus after the injection of Aβ. However, TNFα prevented Aβ‐induced caspase‐3 activation. The results are discussed from the perspective of dual (neuroprotective and neurodestructive) roles of TNF in the brain.

Acknowledgement:  Supported by RBR F (grant # 01‐04‐449477).
HIV‐1 Tat protein disrupts the blood–brain barrier through the oxidative and inflammatory mechanisms
Authors: Toborek M. , Pu H. , Andras I. , Flora G. , Lee Y. W.
Published: May 2003

Tat protein, which is released from HIV‐infected cells, may play a major role in HIV‐induced pathological processes in the CNS, including HIV‐associated dementia. Therefore, the aim of the present study was to examine the effects of Tat on vascular inflammatory responses as well as blood–brain barrier structure and function both in vitro and in vivo environments. Tat treatment induced cellular oxidative processes, activities of redox‐regulated transcription factors, and expression of inflammatory mediators. Most importantly, Tat disrupted the blood–brain barrier and stimulated monocyte infiltration of the CNS. Disruption tight junction protein integrity appeared to be the underlying mechanism of these effects. Among studied junctional proteins, claudin 5 expression was the most affected by Tat treatment. These data provide evidence that Tat can be involved in the blood–brain barrier breakdown and HIV entry into the brain.

Acknowledgements:  Supported by NIH; NS39254, MH63022, and AA013843.
Changes of expression of mitochondrial proteins involved in energy transduction in rat brain during aging
Authors: Nicoletti V. G. , Marino V. M. , Giuffrida Stella A. M.
Published: May 2003

Oxidative damage is the major responsible for mitochondrial dysfunction during aging and many neurodegenerative diseases with respiratory chain Complex I mostly affected. A down‐regulation of the mitochondrial respiratory chain Complex‐I subunits ND1 (mitochondrial encoded) in rat total cerebral cortex RNA and 39 kDa (nuclear encoded) in frontal cortex protein extracts was observed at 6, 12 and 28 months of age. However the 39 kDa subunit showed an age‐related up‐regulation in the parietal cortex and no changes in the cerebellum protein extracts. In frontal and parietal cortex protein extracts a significant reduction of subunit α (nuclear encoded) of FoF1‐ATP synthase, with no changes in the cerebellum, were also observed. A similar impairment was found also for mitochondrial adenine nucleotide translocator ANT‐1 in total rat cerebral cortex RNA. Any change of subunit synthesis might lead to neuronal dysfunction and increase of ROS.
Zinc peptidases and Alzheimer's disease
Authors: Nalivaeva N. N. , Carson J. A. , Fisk L. , Turner A. J.
Published: May 2003

We have investigated the role of zinc peptidases in metabolism of the amyloid precursor protein (APP) and the effects of hypoxia. Two peptidase families have been studied: the neprilysin (NEP) family which includes, in the brain: NEP, endothelin converting enzyme (ECE) and secreted endopeptidase (SEP). Reactive oxygen species can regulate enzyme activity via modulation of the zinc ion at the active site. Both NEP and ECE can prevent accumulation of amyloid beta peptide by hydrolyzing the peptide. As acute and chronic hypoxia can modulate APP processing, we have investigated the effects of hypoxia in cell culture on the expression and activity of NEP, ECE and SEP. In parallel, we have monitored the expression of another zinc peptidase, alpha‐secretase, that mediates the nonamyloidogenic processing of APP. Overall, zinc peptidases appear neuroprotective and modulation of these activities in pathological states could lead to neurodegeneration.

Acknowledgements:  This work was supported by the UK MRC, The Royal Society, INTAS, The Biochemical Society.
Structural studies of Alzheimer's disease Abeta peptide oligomers
Authors: Jabłonowska A. , Bakun M. , Dadlez M.
Published: May 2003

Amyloid beta peptide is recognized as the main constituent of the extracellular amyloid plaques, the major neuropathological hallmark of Alzheimer's disease. Abeta is a small peptide constitutively expressed in normal cells, not toxic in the monomeric form but aggregated Abeta is assumed to be the main if not the only factor causing Alzheimer's disease. Interestingly, the new reports suggest neurotoxicity of soluble Abeta oligomers rather than amyloid fibrils. Because of the fact that fibrils were thought to be the main toxic species in AD, early structural studies focused on fibrils themselves and Abeta monomers, as their building blocks while there is practically no data on oligomer structure and mechanism of neurotoxicity. Using a model peptide spanning residues 10–30 of Abeta, obtained by overexpression in bacteria, we have employed mass spectrometry of noncovalent complexes and disulfide rearrangement assay to gain new insight into structure and dynamics of a prenucleation step of Abeta peptide oligomerisation.
The study of the creatine kinase in rat brain during ischemia by magnetization transfer and biochemical analysis
Authors: Dobrota D. , Likavčanová K. , Kašparová S. , Liptaj T. , Prónayová N. , Mlynárik V. , Belan V.
Published: May 2003

Various methods are used to study the biochemical changes in the central nervous system under normal and pathological conditions. The magnetization transfer 31P magnetic resonance technique was used here to measure the creatine kinase (CK) reaction rate constant in vivo in rats with cerebral ischemia. The measurements indicated that the rate constant of the CK reaction was significantly reduced in the case of chronic brain ischemia in aged rats. The similar reduction of the creatine kinase activity was found in the ischemic rat brain homogenate measured by biochemical analysis. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible or no change in signal intensities of compounds containing macroergic phosphates.

Acknowledgements: This work was supported by the Grant Category C and Comenius University Grant No. X/2003.
In vitro1H magnetic resonance spectroscopy differences between meningeoma and astrocytoma
Authors: Likavčanová K. , Dobrota D. , Liptaj T. , Prónayová N. , Galanda M. , Belan V.
Published: May 2003

Tumor transformation of the human brain cells causes different biochemical changes. Here we employed 1H magnetic resonance spectroscopy to compare the presence of low molecular weight metabolites in meningeoma and astrocytoma tumors by measuring perchloric acid extracts of the cells. In 1H spectra of meningeoma we detected high signal from lactate but were unable to detect any signal of NAA and creatine. In contrast, astrocytoma samples revealed significantly higher level of inositol and glycine and significant decrease in glutamate and glutamine compared with meningeoma but no presence of taurine. Our results suggest that 1H MRS can provide useful information about biochemical changes in different types of brain tumors.

Acknowledgements: This work was supported by the Grant Category C and Comenius University Grant No. X/2003.
High‐efficiency transfection of human endothelial cells mediated by cationic lipids
Authors: Kaiser S. , Toborek M.
Published: May 2003

Cellular transfections provide powerful experimental tools to study gene regulation. However, endothelial cells are difficult to transfect. Therefore, the present studies were performed to optimize transfection techniques in human endothelial cells. To study transfection rates, endothelial cells were transfected with the pGL3 vector, containing the luciferase reporter gene, complexed with several currently available liposomes, such as different Perfect Lipid (pFx) mixtures, DMRIE‐C, or lipofectin. The optimal transfection rate was achieved in cells transfected for 1.5 h with 5 mg/mL of DNA plasmid in the presence of 36 mg/mL of pFx‐7. Transfections mediated by other liposomes were less efficient. Usefulness of the optimized transfection technique was confirmed in cells transfected with NF‐kB or AP‐1‐responsive constructs. We conclude that among several currently available liposomes, pFx‐7 appears to be the most suitable for transfections of cultured human endothelial cells.

Acknowledgements: Supported by NIH; NS39254, MH63022, and AA013843.
Determination of trace elements in Parkinson's diseased brain tissue using microbeam of synchrotron radiation
Authors: Szczerbowska‐Boruchowska M. , Lankosz M. , Adamek D. , Ostachowicz J. , Krygowska‐Wajs A. , Szczudlik A. , Bohic S. , Simionovici A. , Chwiej J.
Published: May 2003

The microbeam synchrotron X‐ray fluorescence (MSXRF) technique is powerful tool for determining elemental composition and spatial distribution of elements within materials on the microscopic level. Therefore, application of this technique seems to be helpful for investigation of the role of trace elements in neurodegenerative processes. As a preliminary study the MSXRF technique was applied for elemental microanalysis in cryo‐sections of substantia nigra tissue for cases of Parkinson's disease (PD) and the control. Two‐dimensional maps of elemental distribution showed significant topographic and quantitative differences between the pathological and control tissues. Quite interesting is significant increased accumulation of Ca, Cu, Fe, Fe, and Zn that was observed in PD neuron perikaryal parts.
Morphological evidence of the continuum between necrosis and apoptosis in model of anoxia in vitro
Authors: Nagańska E. , Matyja E. , Grzywaczewska E.
Published: May 2003

Growing evidence suggests that two modes of cell death, known as apoptosis and necrosis, are involved in postanoxic injury. The current opinion on these two types of cell death is that apoptosis and necrosis are not always the uniform and distinct events. The aim of this study was to determine ultrastructural criteria of postanoxic neuronal changes in model of anoxia in vitro. The organotypic cultures of rat hippocampus exposed to 10‐ and 20‐min of anoxic insult revealed the morphological features classic for both necrotic and apoptotic neuronal cell injury. Some neurones exhibited the typical necrotic lysis whereas others clearly reflected an active apoptotic form of cell death consisting of nuclear condensation with early preservation of cell membranes. However, numerous damaged cells shared both apoptotic and necrotic ultrastructural characteristics. These results evidenced the morphological continuum between apoptosis and necrosis under anoxia in vitro.
DNA damage, p53 gene expression and p53 protein level in the rat brain aging
Authors: Dorszewska J. , Adamczewska‐Goncerzewicz Z.
Published: May 2003

The aging induces free radicals leading to DNA damage (8‐oxo‐2′‐deoxyguanosine, 8‐oxo2dG). DNA injury causes increased expression of p53 gene and p53 protein. Levels of 8‐oxo2dG (HPLC), p53 mRNA (PCR) and p53 protein (Western blot) were estimated in gray matter (GM), white matter (WM), cerebellum (C) and medulla oblongata (MO) of control, 12‐ and 24‐month‐old rats. The level of 8‐oxo2dG increased with age in C (P < 0.05 in 12‐month‐old and P < 0.01 in 24‐month‐old rats) and MO. In 12‐month‐old animals the level of 8‐oxo2dG in GM and WM was higher than in controls. In 12‐month‐old animals p53 gene expression decreased while amounts of p53 protein increased, depending on the oxidative DNA damage. In 24‐month‐old rats, expression of p53 increased in all structures (P ≤ 0.05) while p53 protein showed decreased levels in most of structures of central nervous system (WM, C, MO). Aging leads to increased 8‐oxo2dG and augmented p53 gene expression, accompanied by a lowered expression of p53 protein.
Nicotine up‐regulates expression of neurotrophic factors and attenuates apoptosis of spinal cord neurons
Authors: Garrido R. , Toborek M.
Published: May 2003

Nicotine may induce neuroprotection in spinal cord injury; however, the mechanisms of these effects are not fully understood. The present study focused on the effects of nicotine on expression of neurotrophic factors, such as brain‐derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF‐2) in a model of spinal cord injury. In this model, cultured spinal cord neurons are exposed to 10 mm arachidonic acid (AA). Exposure to AA diminished expression of BDNF and FGF‐2; however, pretreatment with nicotine protected against these effects. Mecamylamine and α‐bungarotoxin inhibited nicotine‐mediated up‐regulation of BDNF and FGF‐2. Moreover, nicotine, BDNF and FGF‐2 protected against AA‐induced apoptosis of spinal cord neurons. These results suggest that AA can induce apoptosis of spinal cord neurons by depletion of neurotrophic factors and that nicotine can protect against these effects through the alpha7 receptor‐mediated pathway.

Acknowledgements:  Supported by grants from Philip Morris Research Program and KSCHIRT.
Vinpocetine exerts cytoprotective activity and prevents apoptosis of astrocytes during in vitro ischemia and reoxygenation
Authors: Gabryel B. , Malecki A. , Herman Z. S.
Published: May 2003

The aim of study was to assess whether vinpocetine (VINP) has protective effect on astrocytes in ischemic injury. Primary cultures of rat astrocytes were deprived of glucose and serum and incubated for 24 h in 3% O2 with 0.1 and 10 μm of VINP either during 24 h normoxia, 24 h simulated ischemia or 24 h reoxygenation. Cells viability was determined by Live/Dead Assay Kit, LDH release and MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] conversion tests. We measured ATP concentration by bioluminescent method and apoptosis by staining with Hoechst 33342 and caspase‐3 assay. Treatment with 0.1 μm VINP during ischemia and reoxygenation significantly decreased the number of apoptotic cells as well as caspase‐3 activity, and increased ATP level and mitochondrial function. Higher VINP concentration (10 μm) was detrimental during ischemia. Experiments proved cytoprotective effect of 0.1 μm VINP on astrocytes in vitro.
Nonapoptotic functions of ‘apoptotic’ cascades: caspase‐3 is involved in hippocampal synaptic plasticity
Authors: Kudryashov I. , Kudryashova I. , Gulyaeva N.
Published: May 2003

Numerous studies establish caspases as essential mediators of cell death. However, many of the same signals that are involved in neuronal death in both physiological and pathological settings may be also involved in developmental and synaptic plasticity in the nervous system. Here we present the experimental data demonstrating the correlation of caspase‐3 activity with population spike amplitude modification in early postnatal ontogenesis suggesting that caspase‐3 is involved in developmental hippocampal synaptic plasticity not related to apoptosis. We also demonstrate that inhibition of caspase‐3 activity in hippocampal slices time‐dependently blocks NMDA‐dependent long‐term potentiation in CA1 region. These results confirm the involvement of caspase‐3 in modulating synaptic plasticity through the cleavage‐dependent alterations in structural proteins and components of signal transduction systems.

Acknowledgement:  Supported by RBR F (grant # 01‐04‐449476).
Role of cytochrom c and Bad protein in post‐ischemic neuronal death – CsA protection
Authors: Dłużniewska J. , Zabłocka B. , Kozłowska H. , Sarnowska A. , Domańska‐Janik K.
Published: May 2003

We have estimated cytosolic cytochrom c (cCyt c) as a measure of mitochondria permeability (MP) and its inhibition by CsA. In hippocampus of gerbils subjected to 5 min ischemia, cCyt c increased biphasically: transiently at 0.5 h and then steadily at ≥24 h. Involvement of proapoptotic protein Bad in the mechanism of MP after ischemia was studied too. Immediate interarterial infusion of CsA (10 mg/kg) totally inhibited early Cyt c translocation and protected CA1 neurons. This effect disappears with delayed application of the drug. Temporal ability of CsA to block apoptotic signaling and Cyt c translocation was verified on primary cortical neurons in vitro by confocal immuno‐imaging. The results confirm time‐limited effectiveness of CsA to inhibit mitochondrial cyclophilin D connected permeability to Cyt c and to protect hippocampus by relatively narrow ‘window of opportunity’ after ischemic insult.

Acknowledgements:  Supported by PBZ‐KBN‐002/CD/P05/2000 and statutory theme 4.
IL‐1 system in apoptosis of murine hippocampal neurons of dentate gyrus induced by trimethyltin administration
Authors: Fiedorowicz A. , Figiel I. , Zaremba M. , Schliebs R. , Oderfeld‐Nowak B.
Published: May 2003

Selective degeneration of murine hippocampal granule cells was induced in 1‐month old BalbC mice by administration of trimethyltin (TMT) (2.5 mg/kg b.w.). Granule cell death bore the apoptotic features. We detected chromatin condensation, oligonucleosomal DNA fragmentation and caspase‐3 activation. Ribonuclease protection assay showed concomitant increase of interleukin‐1beta (IL‐1beta) and IL‐1 receptor antagonist (IL‐1ra) mRNA in the hippocampus. Moreover, an induction of IL‐1beta immunoreactivity was detected in reactive microglia in the area of neurodegeneration. Western blot showed the intensified synthesis of IL‐1ra in the dentate tissue. Temporal and spatial relation between apoptosis and expression of IL‐1 family proteins suggests the double role of these cytokines. Whereas IL‐1beta is probably engaged in neuronal apoptosis, IL‐1ra is involved in protection of spared granule cells.
Neonatal neurodegeneration after N‐acetyl‐aspartylglutamate (NAAG) administration in rat hippocampus
Authors: Bubeníková V. , Balcar V. J. , Druga R. , Pliss L. , Št'astný F.
Published: May 2003

NAAG, an agonist at Group II metabotropic glutamate receptors and at the N‐methyl‐d‐aspartate (NMDA)‐type of ionotropic glutamate receptor, was infused at 250 nmoles in 0.25 μL of saline into each cerebral ventricle of 12‐day‐old rat pups. Proportion of pycnotic neurons was determined in 100 × 200 μm areas of medial and lateral blades of DG, and in areas CA1, CA3a, b of both dorsal and ventral hippocampus. In some sections we used Fluoro‐JADE‐B staining to visualize degenerating neuronal cell bodies. There was a marked neurodegeneration, particularly in the medial blade of DG, at 24 h after the administration of NAAG but it was lower at the 4‐day time point. No significant damage was observed in 50‐day‐old rat. The results indicate that early postnatal administration of NAAG can cause serious loss of neurons in rat hippocampus.

Acknowledgement:  Supported by LNOOB122 MEYS CR.
Is quinolinic acid involved in etiopathogenesis of schizophrenia?
Authors: Št'astný F. , Tejkalova H. , Lisy V. , Buben´ıková V. , Balcar V. J.
Published: May 2003

Retroviruses have been proposed as one of the infectious agents involved in the pathogenesis of schizophrenia. They can cause neuronal death by endogenous toxin(s), such as quinolinic acid (QUIN), released by virus‐activated brain macrophages/microglia. QUIN is an endogenous excitotoxin, with an affinity at specific NMDA‐R heteromers. We have administered QUIN to 12‐day‐old rat males by infusions of 250 nmol QUIN into each lateral ventricle. On day 50 the rats exhibited a tendency to higher basal acoustic startle responses (120 dB), but their prepulse inhibition (PPI) was deficient at 73, 75 and 80 dB. The deficit in PPI correlated with the neuronal losses and with the decreased receptor binding of [3H]glutamate in lesioned brains. The data provide further evidence that the toxin‐induced neuronal deficit and glutamate‐receptor loss in supra‐pontine brain regions may impair sensorimotor gating in the animal model of schizophrenia.

Acknowledgement:  Supported by IGA MH CR No. NF6031‐3.
Neural cell adhesion molecule (NCAM) as a target for the development of drugs for the treatment of neurodegenerative disorders
Authors: Klementiev B. , Novikova T. , Koehler L. , Berezin V. , Bock E.
Published: May 2003

NCAM plays an essential role in neural development and regeneration. We have recently demonstrated a direct interaction between the fibronectin type III modules (F3) of NCAM and the extracellular part of the FGF receptor and also identified a binding site localized in the second F3 module. The corresponding peptide motif (FGL) activated the FGF receptor, promoted neuritogenesis and protected DA neurons in vitro. In a rat model of A‐beta (amyloid‐beta peptide) induced neurite toxicity, FGL rescued the deficit in learning and improved neuropathology. Thus, small artificial mimetics of NCAM with agonist‐like effects are attractive lead‐candidates for drug development for the treatment of neurodegenerative diseases.
Cerebrospinal fluid nitrite/nitrate predicts poor outcome in patients with subarachnoid hemorrhage (SAH)
Authors: Rejdak K. , Petzold A. , Sharpe M. A. , Kay A. D. , Kerr M. , Stelmasiak Z. , Thompson E. J. , Giovannoni G.
Published: May 2003

The study aimed to relate CSF levels of nitric oxide metabolites and oxyhemoglobin (OxyHb) to the clinical outcome measures in patients after SAH. The study group comprised 10 SAH patients and 10 controls. CSF total nitrite/nitrate (NOx) concentration was measured by a vanadium‐based assay while OxyHb level by spectrophotometry. There was a significant correlation between CSF concentrations of NOx and OxyHb (P = 0.001, r = 0.87). When the impact of bleeding into CSF was considered, patients with good outcome had significantly lower CSF NOx than those with bad outcome (P = 0.007). This study demonstrates that after SAH, CSF NOx levels correlate with OxHb and high CSF NOx predicted poor outcome.
Differences in reversed plasmalemmal Na/Ca exchange activities among neuronal subtypes
Authors: Kiedrowski L.
Published: May 2003

Under pathological conditions, when the Na+ and K+ concentration gradients collapse, plasmalemmal K‐dependent (NCKX) and K‐independent Na/Ca exchangers (NCX) may reverse and contribute to toxic Ca2+ accumulation. Vulnerability to ischemia or excitotoxicity greatly varies among neuronal subtypes and may be related, in part, to differences in reverse NCKX and/or NCX activities. Cultured cerebellar granule cells (CGCs) are more resistant to excitotoxicity than forebrain neurons (FNs). To examine whether NCKX or NCX activities in CGCs differ from those in FNs, cytosolic [Ca2+] transients mediated by reversed NCX and NCKX were monitored. NCKX and NCX reversal was triggered by gramicidin‐induced Na+ influx and K+ efflux. The NCX reversal was inhibited by 10 μm KB‐R7943, and that of NCKX by removal of external K+. In FNs, reversed NCX activity was much more robust than in CGCs, however, activity of reversed NCKX was similar. These data support the idea that vulnerability to excitotoxicity may be linked to reversal of plasmalemmal Na/Ca exchangers.

Acknowledgement:  Supported by NS37390.
IRFI 042 reduces oxidative stress against kainic acid toxicity in the rat brain
Authors: Adamo E. B. , Marini H. , Marini R. , Bellomo M. , Altavilla D. , Calapai G. , Bitto A. , Squadrito F.
Published: May 2003

We investigated if IRFI 042, an analog of vitamin E, protects the brain against oxidative stress induced by intraperitoneal administration of Kainic acid (KA) (10 mg/kg); sham brain injury rats were used as controls. Animals received either IRFI 042 (20 mg/kg) or its vehicle 30 min before KA injection and after 6 h were sacrificed to measure malonildyaldheide (MDA) and glutathione levels (GSH) in the diencephalon. Behavioral changes were also monitored. Intraperitoneal administration of IRFI decreased MDA (micromol/g wet tissue: KA + vehicle = 22.5 ± 4.2; KA + IRFI = 17.1 ± 1; P < 0.005) and prevented GSH loss (nmol/g wet tissue: KA + vehicle = 0.41 ± 0.1; KA + IRFI = 1.86 ± 0.2; P < 0.005) in the diencephalon. The latency of occurrence of behavioral signs increased from 39 ± 1 to 62 ± 6 min in IRFI 042 group. The data suggest that IRFI 042 might protect against KA‐induced oxidative stress.
Biotransformation of D‐panthenol and its action in protection of activation of oxidizing processes in brain
Authors: Moiseenok A. G. , Kanunnikova N. P. , Gurinovich V. A.
Published: May 2003

It is a well known fact that 3H‐panthenol (PL) has a high bioavailability, so we studied its biotransformation and its protective action against lipoperoxide activation in homogenates and mitochondrial‐synaptosomal fraction (11 000 g) of rat brain. The lipoperoxidation was initialized by Fe2+‐ascorbate complex (Fe2+‐Asc). In experiments in vivo, after 30 min, we demonstrated accumulation of intermediate products of CoA biosynthesis – pantothenic acid (PA), phospho‐PA, and phosphopantetheine – in postmitochondrial fraction of brain, by using a HPLC technique. Addition of the PL (10 mm) to brain hemispheres homogenates or mitochondrial‐synaptosomal fraction caused a remarkable reduction of malondialdehyde production. However, 30 min preincubation with the PL, but not with PA, was ineffective. The data obtained may be a reason for a high neuroprotective activity of PL in curing brain diseases with vessel or alcohol‐induced damages.
Total antioxidant capacity of human blood plasma from healthy adults
Authors: Lipińska S. , Gorąca A. , Nowak D.
Published: May 2003

Reactive oxygen species are produced as a consequence of normal aerobic metabolism and could be important causative agents of a number of human diseases. Human serum contains many different antioxidants that may have importance of maintenance of antioxidant screen. Blood samples were obtained from healthy volunteers, at young (20–35 years) and old (36–60 years) age. Plasma was prepared and assayed within 3 h from blood collection and analyzed for the antioxidant capacity. At a low pH a ferric tripyridyltriazine (Fe3+‐TPTZ) complex is reduced to the ferrous (Fe2+) form that causes changes of sample absorbance at 593 nm. The plasma antioxidant capacity of young and old group corresponded to 22.69 ± 0.86 and 17.59 ± 1.17 μmol of Trolox (a water‐soluble analog of vitamin E). Young group had significantly higher ferric reducing/antioxidant power value than old subjects. These suggest that plasma antioxidant capacity declines with volunteers age.

Acknowledgement:  The study was financed by a 502‐11‐809 Med. Univ. grant.
Reactive oxygen species production in synaptosomes is independent of ΔΨm
Authors: Sipos I. , Tretter L. , Adam‐Vizi V.
Published: May 2003

This study addressed whether dissipation of ΔΨm had any influence on ROS generation of in situ neuronal mitochondria. The ROS formation was measured as a release of H2O2 by Amplex Red assay. Complete blockage of Complex I by rotenone or Complex III by antimycin caused enhanced H2O2 release. Dissipation of ΔΨm by FCCP or DNP had no effect on the H2O2 production induced by rotenone or antimycin. Antimycin substantially diminishes the ΔΨm, but part of the membrane potential still maintained by the reverse function of mitochondrial ATP synthase. When antimycin was applied together with oligomycin, ΔΨm was totally dissipated, but the ROS production decreased only by 15%. Rotenone inhibited the antimycin‐induced H2O2 release by 55%, but not eliminated completely. These experiments suggest that in in situ mitochondria of synaptosomes (i) ROS generation because of inhibition of Complex I or III is not dependent on ΔΨm and (ii) when Complex I is completely inhibited electrons entering the respiratory chain distal from rotenone site could fuel the ROS formation.
Free radical production in synaptosomes: the effect of respiratory substrates
Authors: Tretter L. , Sipos I. , Miklos M. , Adam‐Vizi V.
Published: May 2003

We investigated whether reactive oxygen species (ROS) production is different when the metabolism is driven by alternative respiratory substrates (RES) in synaptosomes. ROS production was monitored with the Amplex Red assay and parallel with that aconitase activity was measured, as this enzyme is sensitive to inhibition by ROS. Inactivation of aconitase and ROS production in synaptosomes run parallel when ROS production is induced by inhibition of respiratory complexes. In the presence of either isocitrate, alpha‐ketoglutarate (α‐KG), succinate or pyruvate but not citrate ROS production was enhanced as indicated by the Amplex Red assay. However, aconitase activity decreased only when α‐KG was used as a substrate. With other RES aconitase activity was not decreased in spite of enhanced ROS formation. We conclude that (i) in the presence of different RES the amount of ROS production is different; (ii) aconitase assay as a measure of ROS production has a limitation when various RES drive the metabolism.
Mechanisms of neuroprotection in hippocampal organotypic culture
Authors: Sarnowska A. , Kozłowska H. , Domańska‐Janik K.
Published: May 2003

Hippocampal organotypic culture is one of the best suited experimental settings for study neuroprotection in vitro. Here two different models of selective neuronal death in CA1 sector has been investigated: (i) induced by reactive oxygen species after treatment with 30 μm tert‐butylhydroperoxide (TBH); (ii) by glutamatergic receptor/channel agonist 100 μm NMDA. The rate of cell death was monitored at 6 and 24 h post‐treatment after staining of living cells with propidium iodide. Different levels of neuroprotection was observed with: cyclosporine A (a nonspecific ligand of cyclophylins A & D); FK506 (an analog reacting exclusively with cyclophilin A); MK801 (a noncompetitive NMDA‐R antagonist); diazepam (hyperpolarizing agent blocking GABAA‐R); the IGF and its splicing variant MGF (supposed endogenous neurotrophins). Comparison of the drug efficiencies for neuroprotection being observed in these two models may help in better understanding of mechanisms of their action and suggests optimal neuroprotective strategies in related brain pathologies.
Differential expression of heme oxygenase isoforms in rat brain by endotoxin (LPS)
Authors: Calabrese V. , Scapagnini G. , Ravagna P. , Colombrita C. , Spadaro F. , Guagliano E. , Abraham N. G. , Alkon D. L. , Giuffrida Stella A. M.
Published: May 2003

Heme oxygenase‐1 (HO‐1) is a stress protein expressed in various pathological conditions associated with oxidative stress. Brain HO‐1 expression and activity in response to LPS treatment showed regional variability with the highest levels in the substantia nigra (SN) and hippocampus. HO‐1 induction by LPS was redox‐sensitive and associated with increased levels of NO synthase and arginase, two proteins involved in the regulation of cellular redox state. Brain HO‐2 and HO‐3 expression, studied by quantitative RT‐PCR, did not show significant changes. Our data suggest an interaction between NO and the HO system in the brain after LPS treatment. As SN and hippocampus are involved in Parkinson's and Alzheimer's diseases, understanding interaction of these proteins in the brain will help to elucidate the mechanisms involved in neurodegeneration.
Macrophage migration inhibitor factor (MIF) can induce oxidative injury and apoptotic cell death of spinal cord neurons
Authors: Toborek M. , Chalimoniuk M.
Published: May 2003

MIF is a cytokine produced by a variety of cells and tissues including the CNS. It can exert a variety of biological functions, such as induction of inflammatory responses and counterregulation of glucocorticoid effects. However, the role of MIF in the pathogenesis of spinal cord trauma is not fully understood. Therefore, the aim of the present study was to evaluate the cellular effects of MIF in cultured spinal cord neurons. A 3‐h exposure to MIF significantly enhanced intracellular calcium levels; an effect which was prevented by TMB‐8, an antagonist of the IP3 receptor. In addition, MIF treatment increased oxidative stress, decreased viability of spinal cord neurons, increased LDH release and stimulated apoptosis. These results suggest that MIF may play an important role in secondary spinal cord injury.

Acknowledgements:  Supported by grants from KSCHIRT and Philip Morris External Research Program.