Ronit Galron

Endothelin in Cerebrospinal Fluid and Plasma of Patients in the Early Stage of Ischemic Stroke

BACKGROUND AND PURPOSE Endothelin 1 (ET-1), a highly potent endogenous vasoactive peptide, exerts a sustained vasoconstrictive effect on cerebral vessels. Elevation of ET-1 in plasma has been reported 1 to 3 days after ischemic stroke. Since we assumed that a much faster and more intense response may be observed in the cerebrospinal fluid (CSF) and since an increase in concentration of ET-1 in the CSF may cause constriction of cerebral vessels and eventually influence the neurological outcome, we measured ET-1 values in the CSF within 18 hours of stroke onset and compared the values with those in the plasma. METHODS Twenty-six consecutive patients with acute stroke were clinically evaluated according to the modified Matthew Scale and underwent two repeat CT scans. Within 5 to 18 hours of stroke onset, lumbar puncture and blood samples were concomitantly obtained and tested; ET-1 levels in CSF and plasma of these patients were analyzed by radioimmunoassay and compared with the levels of a control group of patients with no neurological disease. RESULTS The mean CSF concentration of ET-1 in the CSF of stroke patients was 16.06 +/- 4.9 pg/mL, compared with 5.51 +/- 1.47 pg/mL in the control group (P < .001). It was significantly higher in cortical infarcts (mean, 17.7 +/- 4.1 pg/mL) than in subcortical lesions (mean, 10.77 +/- 4.1 pg/mL) (P < .001) and significantly correlated with the volume of the lesion (P = .003). The correlation between ET-1 levels in the CSF and the Matthew Scale score was less significant (P = .05). Plasma ET-1 level was not elevated in any group. CONCLUSIONS ET-1 is found to be significantly elevated in the CSF of stroke patients during the 18 hours after stroke. No elevation was demonstrated in plasma at this time period. ET-1 may be used as an additional indicator of ischemic vascular events in the early diagnosis of stroke. The dissimilarity between the CSF and plasma ET-1 concentrations may lead also to an hypothesis that there is a vasoconstrictive effect on the cerebral vessels or a neuronal effect caused by ET-1 in the mechanism of the progression of brain ischemia.

Muscarinic receptor binding in mouse brain: Regulation by guanine nucleotides

Abstract The effect of guanine nucleotides on the binding properties of muscarinic receptors for cholinergic ligands was studied in several brain regions in mice. In homogenates of medulla-pons and cerebellum, GTP (50 μM) caused and 8 -fold decrease in the binding affinity of agonists but had no effect on antagonist binding. Gpp(NH)p was slightly more potent than GTP. The binding properties of both agonists and antagonists in the cortex and hippocampus were unaltered in the presence of guanine nucleotides. The nucleotide effect on agonists arises mainly from the conversion of high affinity to low affinity binding sites. The possible role of these nucleotides in the mechanism of action of muscarinic receptors is discussed.

Functional endothelin/sarafotoxin receptors in rat heart myocytes: Structure-activity relationships and receptor subtypes

Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) family were characterized in newborn rat heart myocytes using human and rat endothelins (ET-1 and ET-3, respectively), SRTX-b and SRTX-c. Binding studies in intact cells and homogenates revealed significantly higher affinities of ET-1 and SRTX-b than of ET-3 and SRTX-c towards these receptors. This binding profile of ET/SRTX peptides points to their interaction with the receptor subtype designated E-S alpha. All four peptides induced time- and dose-dependent phosphoinositide hydrolysis with the following rank order of potency: ET-1 greater than SRTX-b greater than SRTX-c greater than ET-3. Thus, ET-3 which possesses an intermediate affinity toward the receptor was the least effective with regard to this response. These results confirm and extend our earlier report that the ET/SRTX peptides interact with a newly characterized receptor(s) associated with phosphoinositide metabolism and Ca2+ mobilization. The initiation of inositol phosphate formation is largely independent of extracellular Ca2+, verapamil and nifedipine, indicating that the ET/SRTX peptides are not agonists for the voltage-dependent Ca2+-channels.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Brain-machine interfaces (BMI) were born to control “actions from thoughts” in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI—a neuromorphic chip for brain repair—to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary “bottom-up” approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of “finite size networks” which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

γ-Secretase component presenilin is important for microglia β-amyloid clearance.

The cleavage of amyloid precursor protein by γ‐secretase is an important aspect of the pathogenesis of Alzheimers disease. γ‐Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ‐secretase catalytic site. Our aim was to investigate whether γ‐secretase can be important for microglial phagocytosis of Alzheimers disease β‐amyloid.

Endothelin/sarafotoxin receptor heterogeneity: Evidence for different glycosylation in receptors from different tissues

Neuraminidase was used in an attempt to determine whether the endothelin (ET)/sarafotoxin (SRTX) receptor subtypes are glycoproteins and, if so, to determine the role of the carbohydrate moiety in the binding of ligands to the receptor. Incubation of rat cerebellar membranes with neuraminidase was accompanied by a decrease in the capacity of the receptors to bind ET-1 and SRTX-b. In contrast, treatment of the rat caudate putamen and strium or of guinea pig ileum with the enzyme did not affect the binding properties of these receptors. Following exposure of [125I]-ET-1 affinity-labeled receptor to neuraminidase, gel electrophoresis and autoradiography revealed a decrease in molecular mass in the cerebellar and atrial preparations of about 2.5-2.8 kDa. These data indicate that some of the ET/SRTX receptors are glycoproteins and that the sugar moiety is important for ligand binding. Thus, glycosylation might be responsible for the observed heterogeneity among ET/SRTX receptors.

Endothelin/sarafotoxin receptor induced phosphoinositide turnover: Effects of pertussis and cholera toxins and of phorbol ester

The induction of phosphoinositide hydrolysis (PI) by endothelin/sarafotoxin (ET/SRTX) receptors in rat heart myocytes was investigated by the use of bacterial toxins as well as a phorbol ester. Both pertussis- and choleratoxin enhanced the stimulation of PI hydrolysis. Phorbol ester treatment of the myocytes for short periods distinguished between two types of PI-hydrolysis, the one induced by endothelins and the other by sarafotoxins. The possible mediation of G-protein (s) in the induction by ET/SRTX receptors of PI-hydrolysis is discussed.

Contractile effects and binding properties of endothellns/sarafotoxins in the guinea pig ileum

Seven of the eight known isopeptides of the endothelin/sarafotoxin (ET/SRTX) family were tested on the isolated guinea pig ileum and found to cause a concentration-dependent increase in basal tone. The rate or the amplitude of the spontaneous rhythmic contractions of the ileal smooth muscle were essentially not affected by any of the peptides. The maximum contraction elicited by vasoactive intestinal contractor (VIC) was slightly stronger than that induced by endothelin-1 (ET-1) or sarafotoxin-b (SRTX-b), and significantly stronger than the maximal contractions elicited by sarafotoxin-a (SRTX-a), sarafotoxin-c (SRTX-c), or endothelin-3 (ET-3). Sarafotoxin-d (SRTX-d) caused, essentially, no contraction but a rather marked relaxation. The potencies of the various peptides to induce the increase in tension, in terms of EC50 values (cumulative effective concentrations that induce half-maximum response), ranged between 6 and 95 nM depending on the peptide. VIC, ET-1, SRTX-b and SRTX-a had similar potencies and were significantly more potent than SRTX-c and ET-3. A high concentration of SRTX-b elicited no additional response when applied to the organ bath after one of the other peptides had shown a maximal effect. Binding experiments with ileal membranes revealed similar binding properties for the various peptides. Competition with iodinated SRTX-b showed no meaningful differences between the various peptides. It is concluded that all the ET/SRTX peptides compete for the same receptor subtype in the ileum. In terms of efficacy, VIC can be considered as a full agonist of this receptor, SRTX-d is probably an antagonist, while all the other peptides behave as partial agonists.

Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors

Abstract: A study of the effects of bisquaternary pyridinium oximes on calcium‐dependent potassium‐evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]‐acetylcholine release in an atropine‐sensitive and dose‐dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre‐ and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1, 1′‐(4‐hydroxy‐iminopyridinium)trimethylene (TMB‐4) (I50= 8 μM) and the weakest were 1‐(2‐hydroxyiminoethylpyridinium) 1‐(3‐cyclohexylcarboxypyridinium) dimethylether (HGG‐42) and 1‐(2‐hydroxyiminoethylpyridinium) 1‐(3‐phenyl‐carboxypyridinium) dimethylether (HGG‐12) (I50= 150 μM). As postsynaptic antagonists, the latter drugs are more potent (K1= 1.3‐3.3 μM) than TMB‐4 (K1= 50 μM). Combined therapy with two drugs such as TMB‐4 and HGG‐12 might be effective in blocking severe hyper‐activity of the cholinergic system.

MRI evidence of white matter damage in a mouse model of Nijmegen breakage syndrome

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of NBS1, Nbn, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. In this study we used MRI to study the brain morphology and organization of Nbn deleted mice. Using conventional T(2)-weighted magnetic resonance, we found that the brains of the mutant mice (Nbs1-CNS-del) were significantly smaller than those of the wild-type animals, with marked mal-development of the cerebellum. Region of interest analysis of the T(2) maps revealed significant T(2) increase in the areas of white matter (corpus callosum, internal capsule and midbrain), with minor changes, if any, in gray matter. Diffusion tensor imaging (DTI) data confirmed that fractional anisotropy values were significantly reduced in these areas, mainly due to increased radial diffusivity (water diffusion perpendicular to neuronal fibers). Biochemical analysis showed low and dispersed staining for MBP and GalC in Nbs1-CNS-del brains, indicating defects in myelin formation and oligodendrocyte development. Myelin index and protein levels were significantly reduced in these brains. Our results point to a novel function of Nbs1 in the development and organization of the white matter.