Radionuclide venography vs Tc-99m-RBC equilibrium angiography: A comparative paired study

Combined direct injection venography (DIV) and equilibrium angiography (EA) were performed in 13 patients, by injecting in vitro labeled ^99mTc autologous RBC, via pedal veins, and imaging the first pass and the equilibrium phase. This paired comparative study of DIV with EA shows that DIV has advantages over EA because it provides selective information on the deep venous system from the calves to the inferior vena cava. DIV provides information on the flow dynamics and the high contrast first pass images provide better definition of non filling venous segments as well as visualization of collaterals. When using ^99mTc-MAA, lung perfusion can also be imaged. DIV is recommended as the procedure of choice for the diagnosis of DVT and EA should be employed only when pedal vein injection is not possible.     

Early-life epileptiform discharges exert both rapid and long-lasting effects on AMPAR subunit composition and distribution in developing neurons

The perinatal period of brain is characterized by dynamic changes in structure and high propensity for epilepsy. Animal models have shown that alterations of AMPA receptor (AMPAR) assembly or function may be related to seizure-induced cell damage, long-lasting impairments in brain development and seizure threshold. However, effects of earlier epileptiform discharges on AMPAR composition and sub-cellular distribution remain understudied. In this study, we analyzed age-dependent variation of relative GluR1 and GluR2 protein levels in primary cultured rat cortical neurons at 7 DIV, 12 DIV, 17 DIV and 21 DIV. By inducing a single event of epileptiform activity at 6 DIV, we tested the effects of early-life seizure-like insults on AMPAR subunit distribution. We found a significant increase in synaptosomal membrane GluR1 expression in magnesium-free (MGF) medium-treated neurons at each time point detected (p < 0.05), while GluR2 expression increased at 7 DIV, and declined at 17 DIV and 21 DIV respectively (p < 0.05). That is, a trend of high GluR1 with much lower GluR2 expression on the surface membrane of epileptiform discharges experienced neurons over time in culture was presented. These findings in an in vitro model of early-life seizure may inform rodent models of epilepsy, as well as the cellular mechanism involved in epilepsy-associated brain dysfunction.     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.     

Munc13 genotype regulates secretory amyloid precursor protein processing via postsynaptic glutamate receptors

The amyloid precursor protein (APP) can be proteolytically degraded via non-amyloidogenic α-secretase and amyloidogenic β-secretase pathways. Previously, we have identified the presynaptic protein Munc13-1 as a diacylglycerol/phorbolester (DAG/PE) receptor that contributes to secretory, non-amyloidogenic APP processing after PE stimulation. Here, we used organotypic brain slice cultures from wild-type mice and from Munc13-1 knock-out (KO), Munc13-2 KO and Munc13-1/2 double KO (DKO) mice for pharmacological stimulation experiments. First, we demonstrate that neuronal populations and synaptic components important for secretory APP processing develop normally in organotypic brain slice cultures of all genotypes analyzed. Blockade of voltage-gated Na⁺ channels by tetrodotoxin reduced the PE-stimulated secretory APP processing, whereas depolarization by high extracellular K⁺ concentration evoked APP secretion. Additionally, the PE-stimulated APP secretion from Munc13-1 KO brain slices was significantly lower than that from wild-type brain slices. This effect was not observed in brain slices from Munc13-2 KO mice, which is consistent with the lower abundance and subpopulation-specific distribution of Munc13-2 in presynaptic elements. In Munc13-1/2 DKO brain slices, the deficiency of Munc13-1 dominated the effect of APP processing. The Munc13-1 KO effect on APP processing could be rescued by the stimulation of postsynaptic glutamatergic receptors. This indicates that lack of postsynaptic glutamate receptor stimulation in Munc13-1 KO brain slice cultures but not presynaptic mechanisms account for compromised APP processing. We conclude that organotypic brain slices cultures are a valuable tool for studying APP processing pathways in intact neuronal circuits and that neuronal activity is important for maintenance of the non-amyloidogenic APP processing.     

Oligodendrogenesis from neural stem cells: Perspectives for remyelinating strategies

Mobilization of remyelinating cells spontaneously occurs in the adult brain. These cellular resources are specially active after demyelinating episodes in early phases of multiple sclerosis (MS). Indeed, oligodendrocyte precursor cells (OPCs) actively proliferate, migrate to and repopulate the lesioned areas. Ultimately, efficient remyelination is accomplished when new oligodendrocytes reinvest nude neuronal axons, restoring the normal properties of impulse conduction. As the disease progresses this fundamental process fails. Multiple causes seem to contribute to such transient decline, including the failure of OPCs to differentiate and enwrap the vulnerable neuronal axons. Regenerative medicine for MS has been mainly centered on the recruitment of endogenous self-repair mechanisms, or on transplantation approaches. The latter commonly involves grafting of neural precursor cells (NPCs) or neural stem cells (NSCs), with myelinogenic potential, in the injured areas. Both strategies require further understanding of the biology of oligodendrocyte differentiation and remyelination. Indeed, the success of transplantation largely depends on the pre-commitment of transplanted NPCs or NSCs into oligodendroglial cell type, while the endogenous differentiation of OPCs needs to be boosted in chronic stages of the disease. Thus, much effort has been focused on finding molecular targets that drive oligodendrocytes commitment and development. The present review explores several aspects of remyelination that must be considered in the design of a cell-based therapy for MS, and explores more deeply the challenge of fostering oligodendrogenesis. In this regard, we discuss herein a tool developed in our research group useful to search novel oligodendrogenic factors and to study oligodendrocyte differentiation in a time- and cost-saving manner.     

E2F1 is not essential for apoptosis induced by potassium deprivation in cerebellar granule neurons

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentration of potassium. A key regulator of cell cycle, E2F1, was believed to play a role in CGN apoptosis induced by potassium deprivation. However, here we demonstrated that although E2F1 was upregulated in wild type CGNs following potassium deprivation, CGNs that derived from E2F1 knockout mice underwent apoptosis at a similar rate as the wild type. Analysis of the apoptotic neurons revealed no difference in the activation of caspase-3 in E2F1 null and wild type CGNs. Furthermore, knockdown of E2F1 expression by RNA interference failed to attenuate the apoptosis of CGNs induced by potassium deprivation. Taken together, our results suggested that E2F1 is not essential for apoptosis induced by potassium deprivation in CGNs.     

Ammonium accumulation and chemokine decrease in culture media of Gcdh−/− 3D reaggregated brain cell cultures

Glutaric Aciduria type I (GA-I) is caused by mutations in the GCDH gene. Its deficiency results in accumulation of the key metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body tissues and fluids. Present knowledge on the neuropathogenesis of GA-I suggests that GA and 3-OHGA have toxic properties on the developing brain.We analyzed morphological and biochemical features of 3D brain cell aggregates issued from Gcdh^?/? mice at two different developmental stages, day-in-vitro (DIV) 8 and 14, corresponding to the neonatal period and early childhood. We also induced a metabolic stress by exposing the aggregates to 10 mM l-lysine (Lys).Significant amounts of GA and 3-OHGA were detected in Gcdh^?/? aggregates and their culture media. Ammonium was significantly increased in culture media of Gcdh^?/? aggregates at the early developmental stage. Concentrations of GA, 3-OHGA and ammonium increased significantly after exposure to Lys. Gcdh^?/? aggregates manifested morphological alterations of all brain cell types at DIV 8 while at DIV 14 they were only visible after exposure to Lys. Several chemokine levels were significantly decreased in culture media of Gcdh^?/? aggregates at DIV 14 and after exposure to Lys at DIV 8.This new in vitro model for brain damage in GA-I mimics well in vivo conditions. As seen previously in WT aggregates exposed to 3-OHGA, we confirmed a significant ammonium production by immature Gcdh^?/? brain cells. We described for the first time a decrease of chemokines in Gcdh^?/? culture media which might contribute to brain cell injury in GA-I.     

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC₅₀ values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC₅₀ value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC₅₀ values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.     

Characterization of protein kinase C isoforms in primary cultured cerebellar granule cells

Protein kinase C (PKC) is a family of serine/threonine kinases comprised of 10 isoforms. Although commercial antibodies are available for all 10 isoforms, the specificity of these antibodies has been questioned. We have identified immunoblot conditions in which commercially purchased PKC antibodies are specific for their respective isoform. We then used these conditions to determine that PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma, lambda, theta, and zeta are present in rat primary cultured cerebellar granule cells (CGCs) 6-14 days in vitro (DIV). This PKC profile is identical to that observed in cerebellar homogenates taken from 6-, 14- and 21-day-old rats. Western blot analysis indicated that the classical and the atypical PKC isoforms were more prevalent in the cytosolic subcellular fraction compared to the particulate fraction under basal conditions. Immunoreactivity for the novel isoforms tended to be higher in the particulate fraction under basal conditions. Phorbol 12-myristate 13-acetate (PMA) treatment resulted in translocated immunoreactivity from the cytosolic to the particulate fraction for all of the classical and novel PKC isoforms, but not for the atypical isoforms. However, the degree of translocation as well as the speed of translocation varied among the isoforms. The stability of the individual isoforms after PMA-induced activation also varied among the isoforms. Differences in these parameters were dependent upon culture batches and PKC isoform groups. We have identified experimental conditions in which reproducible results can be obtained with primary cultured CGCs in the study of PKC. We discuss possible solutions for problems encountered when utilizing primary cultured neurons to study PKC-mediated signal transduction.