Lumbar spinal stenosis: methods of treatment with emphasis on epidural steroid injections

Background and Purpose: The aim of the study was to compare two techniques of steroid application into epidural space to patients with lumbar spinal stenosis (LSS), a chronic degenerative spine disorder. Patients and Methods: Sixty LSS patients have been distributed into 2 groups: “BLIND” (n=30, interlaminar epidural steroid injection without RTG control) and “RTG” (n=30, transforaminal epidural injection with RTG control). All patients have received 80 mg of triamcinolon (Kenalog) into epidural space on L4/L5 level, together with 0,5% lidocain (patients in RTG group 3 ml and those in BLIND group 10 ml) in 3 week intervals. They were asked to describe the pain using visual analogue scales (VAS) at the beginning of treatment (VAS-0), after the first (VAS-1), the second (VAS-2) and the third epidural injection (VAS-3). The differences between groups were shown using t-test (age) and c2-test (gender). Medians of VAS scores were statistically described using non parametrial methods. P<0.05 was considered as a statistically significant. Results: There is no statistical difference among patients regarding to age (P=0.93), gender (P=0.12) and VAS-0 score before the first injection (P=0.27). There is a statistically significant reduction of pain in relation to VAS-0 in both groups (P<0.001). Both groups do not statistically differ when it comes to their effectiveness in regards to VAS scores. Conclusions: We did not find any statistical difference in postinterventional VAS scores among two groups of patients. Choice of technique depends on the experience of the anesthesiologist, as well as on the local technical possibilities (availibility of RTG devices)

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway

Loss of cell-cell and cell-substrate contacts in single pancreatic β-cells divert insulin release to intracellular vesicular compartments

Cell-cell or cell-substrate interactions are lost when cells are dissociated in culture, or during pathophysiological breakdowns, therefore impairing their structure and polarity, and affecting their function. We show that single rat β-cells, cultured under non-adhesive conditions, form intracytoplasmic vacuoles increasing in number and size over time. We characterized these structures and their implication in β-cell function

Engineering of primary pancreatic islet cell spheroids for three-dimensional culture or transplantation: a methodological comparative study

Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production

Phospho-BAD BH3 Mimicry Protects β Cells and Restores Functional β Cell Mass in Diabetes

Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing β cells will greatly augment β cell replacement therapies in type 1 diabetes (T1D). We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3) domain of BAD impart β-cell-autonomous protective effects in the face of stress stimuli relevant to β cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased β cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of β cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional β cell mass in diabetes

Light Entrains Diurnal Changes in Insulin Sensitivity of Skeletal Muscle via Ventromedial Hypothalamic Neurons

Summary: Loss of synchrony between geophysical time and insulin action predisposes to metabolic diseases. Yet the brain and peripheral pathways linking proper insulin effect to diurnal changes in light-dark and feeding-fasting inputs are poorly understood. Here, we show that the insulin sensitivity of several metabolically relevant tissues fluctuates during the 24 h period. For example, in mice, the insulin sensitivity of skeletal muscle, liver, and adipose tissue is lowest during the light period. Mechanistically, by performing loss- and gain-of-light-action and food-restriction experiments, we demonstrate that SIRT1 in steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamic nucleus (VMH) convey photic inputs to entrain the biochemical and metabolic action of insulin in skeletal muscle. These findings uncover a critical light-SF1-neuron-skeletal-muscle axis that acts to finely tune diurnal changes in insulin sensitivity and reveal a light regulatory mechanism of skeletal muscle function. : Aras et al. provide in vivo evidence that tissue responsiveness to insulin varies in a diurnal fashion. In skeletal muscle, the authors show that photic inputs entrain diurnal changes in clock genes expression and insulin sensitivity via SIRT1 in neurons within the hypothalamic ventromedial nucleus

Rab37 knock-down impairs hormone secretion induced by secretagogues.

<p><b>A</b>. The level of endogenous Rab37 (<b>upper panel</b>) and exogenously expressed Rab37-EGFP (<b>lower panel</b>) were estimated by immunoblotting in cells transfected with control shRNA, shRab37 (1) or (2). Equal loading of the lanes was verified using an antibody against actin. <b>B</b>. To assess hormone release, the cells were transiently co-transfected with a plasmid encoding the human growth hormone and with plasmids driving the expression of shRNAs against GFP (control) or with two different shRNAs against Rab37 (shRab37 (1) or (2)). Three days later, the cells were tested for hormone release. For this, they were maintained at 2 mM glucose, (open bars) and then incubated in stimulating solutions (filled bars) containing 20 mM glucose and the cAMP-raising agents Forskolin and IBMX. The amount of hGH expressed by the cells and released in the medium was quantified by ELISA. Hormone secretion under basal and stimulatory conditions is given as % of hGH cellular content. The results are the means of five independent experiments ± SEM. ** p<0.01, *** p<0.001. One-way ANOVA.</p