Alpha synuclein dimers and oligomers are increased in overexpressing conditions in vitro and in vivo.

Parkinson's disease is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta along with the formation of intracellular fibrillar inclusions (Lewy bodies and Lewy neuritis), which are mainly composed of aggregated α-synuclein (ASYN). This latter is a 14 kDa protein that localizes to synaptic vesicles in nerve terminals and promotes soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly. We explored the monomeric and oligomeric state of ASYN in vitro in HEK293s and SH-SY5Y cell lines. In addition rats were injected in the substantia nigra with an Adeno associated virus carrying the human A53T mutation of ASYN (in vivo experiments). We show that human wild type ASYN as well as PD-linked mutations (A30P, E46K and A53T) in overexpressing conditions mostly exists in a monomeric state in equilibrium with dimeric forms. The monomer/dimer ratio is unaffected by PD-linked mutation. Furthermore, the A30P, E46K and A53T mutations overexpression strongly increased cell death compared to wild type ASYN. Taken together, our data suggest that ASYN dimers amount do not directly correlate to reduced cellular viability, suggesting a different role in protein function and induced pathology. Our data suggest that early ASYN neuro-pathogenic effects are probably mediated by other molecular processes than increased oligomerization alone.

Continuous drug delivery in early- and late-stage Parkinson's disease as a strategy for avoiding dyskinesia induction and expression.

The treatment of the motor symptoms of Parkinson's disease (PD) is dependent on the use of dopamine replacement therapy in the form of L: -dopa and dopamine agonist drugs. However, the development of dyskinesia (chorea, dystonia, athetosis) can become treatment limiting. The initiation of dyskinesia involves a priming process dependent on the presence of nigral dopaminergic cell loss leading to alterations in basal ganglia function that underlie the expression of involuntary movements following the administration of each drug dose. Once established, dyskinesia is difficult to control and it is even more difficult to reverse the priming process. Dyskinesia is more commonly induced by L: -dopa than by dopamine agonist drugs. This has been associated with the short duration of L: -dopa causing pulsatile stimulation of postsynaptic dopamine receptors compared to the longer acting dopamine agonists that cause more continuous stimulation. As a result, the concept of continuous dopaminergic stimulation (CDS) has arisen and has come to dominate the strategy for treatment of early PD. However, CDS has flaws that have led to the general acceptance that continuous drug delivery (CDD) is key to the successful treatment of PD. Studies in both experimental models of PD and in clinical trials have shown CDD to improve efficacy, but reduce dyskinesia induction, and to reverse established involuntary movements. Two key clinical strategies currently address the concept of CDD: (1) in early-, mid- and late-stage PD, transdermal administration of rotigotine provides 24 h of drug delivery; (2) in late-stage PD, the constant intraduodenal administration of L: -dopa is utilized to improve control of motor symptoms and to diminish established dyskinesia. This review examines the rationale for CDD and explores the clinical benefit of using such a strategy for the treatment of patients with PD.

Transcriptional alterations under continuous or pulsatile dopaminergic treatment in dyskinetic rats.

Continuous dopaminergic treatment is considered to prevent or delay the occurrence of dyskinesia in patients with Parkinson's disease (PD). Rotigotine is a non-ergolinic D(3) > D(2) > D(1) dopamine-receptor agonist for the treatment of PD using a transdermal delivery system providing stable plasma levels. We aimed to investigate the differential influence on gene expression of pulsatile L: -DOPA or rotigotine versus a continuous rotigotine treatment. The gene expression profile within the nigro-striatal system of unilateral 6-hydroxydopamine-lesioned rats was assessed in order to differentiate potential changes in gene expression following the various treatment using Affymetrix microarrays and quantitative RT-PCR. The expression of 15 genes in the substantia nigra and of 11 genes in the striatum was altered under pulsatile treatments inducing dyskinetic motor response, but was unchanged under continuous rotigotine treatment that did not cause dyskinetic motor response. The route of administration of a dopaminergic drug is important for the induction or prevention of motor abnormalities and adaptive gene expressions. The decline of neurotrophin-3 expression under pulsatile administration was considered of particular importance.

Eicosanoids in rat brain during ischemia and reperfusion--correlation to DC depolarization.

The effects of complete ischemia on cerebral arachidonic acid (AA) metabolism were investigated in the isolated perfused rat brain. During 12.5 min of ischemia, AA, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid increased 129-, 4-, and 10-fold, respectively, while subsequent reperfusion for 30 min resulted in normalized levels independently of the duration of preceding ischemia. Prostaglandin (PG) F2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 remained at preischemic levels during 12.5 min of complete ischemia. However, at the end of subsequent reperfusion for 30 min, the levels of the prostanoids PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and TxB2 increased according to the preceding ischemic time. The levels reached a maximum after 7.5 min of ischemia and were elevated by 7-, 14-, 48-, 3-, and 30-fold, respectively. A prolongation of ischemia of up to 12.5 min was not associated with further increases of prostanoids at the end of reperfusion. The mechanisms underlying the metabolism of eicosanoids are discussed in relation to the changes of cortical direct current potential.

Clinical utility of monitoring tacrolimus blood concentrations in liver transplant patients.

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme-linked immunosorbent assay (ELISA) for monitoring whole-blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7-day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

The internal reference technique in microdialysis: a practical approach to monitoring dialysis efficiency and to calculating tissue concentration from dialysate samples.

In microdialysis experiments, 'recovery' estimations are required to calculate extracellular concentrations of the compounds determined. Generally, relative recovery (RR) is determined in vitro as: RR = cd/cs, with (cd) being the concentration of a compound in a dialysate fraction and (cs) its known concentration within a sample solution. To determine recoveryin vivo, relative loss (RL) was defined RL = (cp-cd)/cp with (cp-cd) being the loss of a compound from the perfusate and (cp) its perfusate concentration. RL was determined in vitro and in vivo by adding an 'internal reference compound' to the perfusate. Here, 14C-labelled lactate was used as the compound of interest. Comparing RL and RR in vitro, we found both to be similar. In vivo, however, RL was 34% of RL(in) vitro (CSF) and 46% of RL(in) vitro in agar-containing CSF. During ischaemia, RL of lactate even decreased to only 35% of the pre-ischaemic control level. We conclude that RL and RR represent inverse measurements of 'recovery.' Whereas RR can only be determined in vitro, RL can be determined in vivo. We found recoveryin vivo to be different from recoveryin vitro. Moreover, recoveryin vivo decreased during ischaemia. By means of the measured recoveryin vivo extracellular lactate concentrations prior and during ischaemia were calculated. The results, therefore, validate the 'internal reference technique' as a practical method for estimating recoveryin vivo and for controlling dialysis efficacy in vivo even continuously.

Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia.

Generalized neurotransmitter overflow into the extracellular space, after cerebral ischemia, has been suggested to contribute to subsequent neuronal death. This study aims to investigate the striatal release of the neurotransmitters dopamine (DA), glutamate (Glu) and gamma-aminobutyric acid (GABA) by means of microdialysis, in a rat model for focal transient cerebral ischemia. Ischemia was induced by the application of 120 pmol endothelin-1 (Et-1), adjacent to the middle cerebral artery (MCA) in freely moving rats. Ischemia produced a large increase in extracellular striatal DA concentrations (2400%), Glu (5500%) and GABA (800%) concentrations. Laser Doppler flowmetry in anaesthetized rats, indicated that the blood flow within the striatum decreased by 75+/-11%. The period of sustained drop of blood flow, was dose-dependently related to the concentration Et-1 injected. Histological analysis of brain slices, taken from anaesthetized and conscious animals, indicated a 500 pmol dose of Et-1 was required to produce a similar infarct in anaesthetized rats to a 120 pmol dose of Et-1 in freely moving rats. The immediate drop in striatal blood flow, and the prompt increase of extracellular DA, after the micro-application of Et-1, were quite striking. This suggests that the DA release, rather than the Glu overflow may be the primary event initiating the cascade of processes ultimately leading to cell death and neurological deficits.

Self-sustained spreading depressions in the chicken retina and short-term neuronal-glial interactions within the gray matter neuropil.

The chicken retina is an accessible piece of intact gray matter in which a self-sustained form of the 'Spreading Depression' (SD) wave can be easily elicited and recorded for many hours with double barrel ion-sensitive electrodes in the extracellular space. The blockade of glial (Müller) cell potassium channels with barium chloride added to the perfusing Ringer depressed both the negative potential shift typical of SDs and the velocity of spread. Moreover, there was separation of the extracellular increase of potassium and the drop in the extracellular potential: the peak of the potassium wave was increased, as well as its duration whereas the potential wave could be depressed to zero or even inverted to positive. By contrast the transient extracellular calcium drop could not be separated from the extracellular potential wave but appeared related to it: no transient calcium drop was observed when the negative potential was completely depressed or inverted. Both, the amplitude of the extracellular potential and extracellular calcium activity appeared to be important factors controlling the velocity of spread.

Spreading depression in human neocortical slices.

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Lubeluzole blocks increases in extracellular glutamate and taurine in the peri-infarct zone in rats.

A microdialysis probe was positioned inside the peri-infarct zone of a photochemically induced neocortical infarct in rats. Extracellular glutamate rose within 20 min after the start of infarct induction and continued to increase during the 5 h observation period to 5.5-fold the pre-infarct baseline value of 0.8 +/- 0.4 micromol/l. Glutamine increased only 1.4-fold. Changes in peri-infarct glutamate were preceded by steep rises in taurine (a 3.9-fold increase from the baseline value of 2.8 +/- 0.7 micromol/l), which coincided with spreading depressions during infarct induction. Post-treatment with lubeluzole ((S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoro-phenoxy) methyl]-1-piperidineethanol, 1.25 mg/kg i.v.), a new cerebroprotective drug, blocked the peri-infarct increases of glutamate and taurine, whereas the R-enantiomer was ineffective. Since lubeluzole has previously been shown to stereospecifically decrease glutamate-activated nitric oxide (NO) toxicity in vitro, the present in vivo stereospecific effect of lubeluzole may be related to modulation of the cascade of NO toxicity, thus preventing NO toxicity-mediated increases in extracellular glutamate. Blockade of the peri-infarct taurine response suggests that lubeluzole also may have reduced cellular osmotic stress in the peri-infarct zone.

Lactate and pH change in close correlation in the extracellular space of the rat brain during cortical spreading depression.

pH sensitive microcelectrodes were used in combination with microdialysis (MD) technique to measure extracellular pH (pHe) and extracellular lactate (lace) within the cortex of rat brains during cortical spreading depression (SD). SD was induced by local K(+)-application and identified by DC recordings. It was accompanied by an extracellular acidification of 0.34 +/- 0.06 pH units and by a 2.8 +/- 0.80 fold increase of lace; the recovery of pHe took place within three phases, that of lace within 2 phases. The recovery of both parameters was complete about 45 min after the onset of SD. We conclude that the changes of lace and pHe are closely related. This indicates both lactate and protons to be transported in parallel.

Extracellular changes of inorganic phosphate are different during spreading depression and global cerebral ischemia of rats.

Tissue levels of inorganic phosphate (iP-) and lactate (lac) increase during cerebral ischemia and cortical spreading depression (SD). Since cell membranes become leaky during these insults, iP- and lac were expected to leak into the extracellular space (ECS). In order to find out whether this occurs or does not, a microdialysis (MD) fiber was implanted into the cortex of anesthetized rats and extracellular lactate (lac(e)) and extracellular iP- (iPe-) were determined during various insults. Extracellular lactate increased to about the same extent during ischemia and SD. In contrast, iPe- increased during ischemia but not during SD. Instead, iPe- started to rise after SD and reached its maximum about 45 min later. The distinct pattern of iPe- in comparison to lac(e) during the above mentioned insults points to a qualitative difference of the underlying mechanisms: whereas lac appears within the ECS at any stressful situation, elevation of iP- within the ECS indicates depletion of energy stores in parallel to the lack of control of ion homeostasis.

Oxygen consumption and energy state in an in vitro model of rat brain perfused with artificial oxygen carrier.

The present study was performed in order to determine the oxygen consumption of a rat brain perfused with an artificial oxygen carrier. Measurements were performed prior to and 2 or 30 min after an ischemic period of 5 min. In addition, the energy-related metabolites were determined. Basal oxygen consumption and energy state were comparable to in vivo conditions. The tissue concentration of the energy metabolites decreased during ischemia and completely recovered during 30 min of reperfusion. The oxygen consumption was higher in the early phase of reperfusion than under pre-ischemic conditions. However, the oxygen consumption in the later phase of reperfusion was lower than the basal consumption. The data demonstrate that the addition of an artificial oxygen carrier to the perfusate provides sufficient amounts of oxygen to the in vitro preparation and that the measurement of the oxygen consumption during the post-ischemic reperfusion is a more sensitive parameter for the detection of emerging deficits than the measurement of the tissue levels of the energy metabolites.

Long-term effects of postnatal hypoxia and flunarizine on the dopaminergic system.

Long-term changes of learning behavior and of the striatal dopaminergic system were observed in a rat model of early postnatal hypoxia. Striatal dopamine (DA) concentration, K(+)-stimulated DA release from slices, and DA uptake into crude synaptosomal preparations (S1 fractions) were used as markers of the striatal DAergic system. Active avoidance learning was tested as behavioral criterion. Cyclodextrin and flunarizine were found to produce long-term effects on the DAergic system in control animals. While cyclodextrin normalized hypoxia-induced effects in DA release, flunarizine prevented those in DA uptake and improved avoidance learning.

[Prospective comparison of hydrosonography, endosonography and specimen sonography for TN staging of gastric carcinoma]. / Prospektiver Vergleich von Hydrosonographie, Endosonographie und Präparatesonographie beim TN-Staging von Magenkarzinomen.

PURPOSE: To compare hydrosonography (HUS), endosonography (EUS) and experimental sonography (PUS) with respect to TN-staging accuracy of gastric carcinoma. MATERIAL AND METHODS: Thirty-six patients with gastric carcinoma underwent EUS (7.5/12 MHz transducer, Olympus GF-UM 20) and HUS (3.75 MHz transducer, Toshiba, Sonolayer SSA-270A) for TN-staging according to the UICC-classification. The resected specimens were reexamined (3.75/7.5 MHz transducer) and again TN-staging was performed. Findings were correlated with histopathological results. RESULTS: T- and N-staging accuracies were as follows: EUS 54 % (19/35) and 79 % (27/34); HUS 41 % (15/37) and 61 % (22/36); and PUS 51 % (19/37) and 72 %(26/36). Sensitivities and specificities for the detection of lymph node metastases were as follows: EUS 87 % and 54 %; HUS 57 % and 69 %; and PUS 83 % and 54 %. CONCLUSIONS: The accuracy of sonographic TN- staging is limited in patients with gastric carcinoma. Nevertheless, EUS may contribute to the preoperative management of patients with gastric carcinoma if indications are well defined. HUS is not suited for TN-staging of gastric carcinoma.

Extracellular taurine as a parameter to monitor cerebral insults 'on-line': time courses and mechanisms as studied in vivo.

UNLABELLED: Taurine increases in the zone surrounding a thrombotic infarct which could be prevented by a neuroprotective drug. Therefore, we aimed at studying the possible release mechanisms since the monitoring of taurine might give valuable information on the progress of cerebral insults and the effect of drugs. A microdialysis membrane was implanted into the cortex of anaesthetised rats: As toxic triggers possibly released by the dying cells in the peri-infarct zone, either a glutamatergic agonist (NMDA) or high potassium were applied via the microdialysis probe. Alternatively, a diluted perfusate was applied to induce cell swelling directly. NMDA antagonists or the NO synthase inhibitor L-NAME were applied locally too. NMDA, NO, high potassium or the hypotonic solution stimulated the release of taurine. The effect of high potassium could be prevented by Ketamine, but not by APV. The effect of NMDA could be inhibited by APV or Ketamine or the NO synthase inhibitor L-NAME. The release of taurine induced by the hypotonic solution could not be be reduced by any of the inhibitors. These data suggest that the release of taurine induced by glutamatergic activity is mediated via the NO cascade. The potassium mediated release seems to be related only in part to glutamatergic activity. Thus, other mechanisms seem to be predominate in potassium mediated swelling. Hypoosmotically induced taurine release is not mediated via the NO cascade and also seems to differ from the aforementioned release mechanisms. IN CONCLUSION: Monitoring of extracellular taurine allows to follow pathological events and to differentiate drug effects.

The measurement of extracellular inorganic phosphate gives a more reliable indication for severe impairment of cerebral cell function and cell death than the measurement of extracellular lactate.

The measurement of cerebral extracellular lactate levels has been suggested to be used to monitor cerebral function in intensive care However, although an increase of extracellular lactate levels is a sensitive parameter for increased cellular activity in general, it will be shown that its prognostic value is limited in regard to the severity of the impairment of cellular function. As an alternative the measurement of the extracellular levels of inorganic phosphate (IP) or adenosine is proposed here: Whereas extracellular lactate levels increased rapidly to about the same extents during ischemia (IS) and spreading depression (SD), IP rose during IS only. Adenosine, on the other hand, increased during both events to a different degree. If, therefore, lactate was the only parameter to be monitored after a cerebral insult, the results would not allow to discriminate between a transient, spontaneously recovering event as a SD and a long lasting or an irreversible loss of cell function as in persisting ischemia/hypoxia. The measurement of IP, therefore, seems to be more suitable than that of lactate or adenosine since IP will appear within the extracellular space only after a sustained failure of membrane function. Thus, the measurement of IP changes turned out to be the more useful parameter for intensive care supervision.

Extracellular changes of taurine in the peri-infarct zone: effect of lubeluzole.

Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in a photochemical stroke model in rats. Tissue swelling, which occurs in the peri-infarct zone, is accompanied by a compensatory taurine release. Therefore, using a microdialysis technique, we aimed at measuring changes of extracellular concentrations of taurine in the peri-infarct zone and the effects of lubeluzole and its R-isomer. Lubeluzole blocked the increase of taurine in tissue immediately surrounding a photochemically induced thrombotic neocortical infarct. By contrast, the R-isomer was completely inactive. We hypothesize that lubeluzole may reduce osmoregulatory stress in peri-infarct tissue.

Study on the cerebral effects of sabeluzole.

The cerebral effects of subeluzole have been studied using the following methods: hypobaric hypoxia in mice, complete ischemia by decapitation in mice, anoxic hypoxia in mice, hemic hypoxia in rats, incomplete ischemia by bilateral carotid ligation in rats, anoxic hypoxia in rats and asphyxic hypoxia in cats. Sabeluzole was active in all the models used: it increased the survival time in hypobaric hypoxia (maximum at 40 mg/kg--by 92.0%, p less than 0.001), survival time in anoxic hypoxia in mice (maximum at 40 mg/kg--by 27.2%, p less than 0.001), gasping in decapitation model (maximum at 20 mg/kg--by 155.4%, p less than 0.001) and survival in hemic hypoxia (maximum at 2.5 mg/kg--by 21.1%, p less than 0.05). The duration of the effect as evaluated in the decapitation model was about 6 h. In incomplete ischemia in rats, however, it showed a weak effect. In anoxic hypoxia in rats, sabeluzole (5 mg/kg i.v.) increased the time latency between onset of anoxia and negative DC-shift by 20.5% and the K+e-threshold by 25.7%. In asphyxic hypoxia in cats, sabeluzole (0.5 mg/kg i.v.) counteracted the hypoxia-induced decrease of the fast-wave amplitudes during the cortical resistance period and the hypoxia-induced decrease of the slow-wave and increase of the fast wave amplitudes during the cortical recovery period.

[Redox sensitive behavior of selenite in the presence of reactive oxygen species. Are there nonenzymatic direct reaction pathways]. / Zum redoxsensitiven Verhalten von Selenit in Gegenwart reaktiver Sauerstoffspezies. Gibt es nichtenzymatische direkte Reaktionswege?

From extensive research over the last decade it has been known that selenium is essential as necessary component of selenoaminoacids and of specific enzymes. Among others, the redoxpair GSH/GSSG is closely connected with antioxidative processes. Moreover it inhibits and/or activates molecular key reactions with the involvement of various small reactive O- and N-species. We investigated the direct interaction of selenite with components of the respiratory burst of human blood cells, considering the redoxamphoterie of alkali-selenite. Selenite tend to redox-disproportation depending on the pH-value. Whether selenite leads to oxidation or reductation is dependent not only on the pH-value, but also on the redox-potential of the reaction partners. In in-vivo adapted in-vitro conditions (ph = 7.4; mumolar concentrations of reaction partners) we observed the following results: 1. SeO3(2-) is not oxidized by H2O2/NO or triplet-oxygen, when the oxidatives are applied alone; 2. SeO3(2-) is quantitatively oxidized from SeO4(2-) by the combination H2O2/NO2- or O2-/NO; 3. SeO3(2-) is semiquantitatively oxidized by singlett oxygen to SeO4(2-). The composition of reaction products was measured by 77Se-NMR-spectroscopy. The reactive intermediate product for the 2. reaction should be peroxynitrite (HOONO). One cannot rule out the possibility that HOONO reacts on a large scale with H2O2 to singlett oxygen. Subsequently singlett oxygen oxidizes selenite. The pathophysiological impact of singlett oxygen in processes like arteriosclerosis is now being investigated. It has been supposed, that singlett oxygen is participating in processes of lipidperoxidation invivo. Further investigations have to show, to what extent selenite is effective as direct 1O2-scavanger.