Seated Position Does Not Change Lumbar Dimensions Compared With Lateral Position.

OBJECTIVE: The infant lumbar puncture (LP) can be a technically challenging procedure. Understanding the anatomical lumbar dimensions may optimize LP conditions. Data from preterm neonates, older children, and adults indicate measurements of the lumbar spine in the seated LP position may be superior when compared with the lateral position. We use point-of-care ultrasound (US) to determine if the seated position, when compared with the lateral decubitus position, significantly affected the lumbar dimensions of infants 12 months or younger presenting to the pediatric emergency department. METHODS: We conducted a prospective observational study of a convenience sample of patients 12 months or younger. We used US to obtain 3 still images oriented longitudinally in the midline over the L3 to L4 interspace in the lateral decubitus and seated positions. A US fellowship-trained emergency physician, blinded to patient position, measured interspinous space, subarachnoid space width, and spinal canal depth. We then compared the means of all 3 dimensions in the lateral and seated positions. RESULTS: From 50 subjects, 49 subjects provided 46 evaluable sets of images for each measure. Interspinous space, spinal canal depth, and subarachnoid space width did not differ significantly between positions. Mean differences did not exceed 0.02 cm for any of the measured dimensions. We report no significant differences in the 3 lumbar dimensions at the seated position when compared with the lateral decubitus position. CONCLUSIONS: For infants younger than 12 months, sonographic measurements of lumbar dimensions did not differ between the positions commonly used for LP.

Effects of the abused inhalant toluene on ethanol-sensitive potassium channels expressed in oocytes.

Toluene (methylbenzene) is representative of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this laboratory and others have shown that these compounds alter the function of a variety of ion channels including ligand-gated channels activated by ATP, acetylcholine, GABA, glutamate and serotonin, as well as voltage-dependent sodium and calcium channels. It is less clear what effects toluene may have on potassium channels that act to reduce the excitability of most cells. Previous studies have shown that ethanol potentiates the function of both the large conductance, calcium-activated potassium channel (BK) and specific members of the G-protein-coupled inwardly rectifying potassium channels (GirKs). Since toluene and other abused inhalants share many behavioral effects with ethanol, it was hypothesized that toluene would also enhance the function of these channels. This hypothesis was tested using two-electrode voltage-clamp electrophysiology to measure the activity of BK and GirK potassium channel currents expressed in Xenopus laevis oocytes. As reported previously, ethanol potentiated currents in oocytes expressing either BK or GirK2 channels. In contrast, toluene caused a concentration-dependent inhibition of BK channel currents with 3 mM producing approximately 50% inhibition of control currents. Currents in oocytes injected with GirK2 mRNA were also inhibited by toluene while those expressing GirK1/2 and 1/4 channels were minimally affected. In oocytes co-injected with mRNA for GirK2 and the mGluR1a metabotropic receptor, exposure to glutamate potentiated currents evoked by a high-potassium solution. Toluene inhibited these glutamate-activated currents to approximately the same degree as those induced under basal conditions. The results of these studies show that toluene has effects on BK and GirK channels that are opposite to those of ethanol, suggesting that these channels are unlikely to underlie behaviors that these two drugs of abuse share.

Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx.

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.

Inhibition of gap junction currents by the abused solvent toluene.

Abused inhalants are a large class of compounds that are inhaled for their intoxicating and mood altering effects. They include chemicals with known therapeutic uses such as anesthetic gases as well as volatile organic solvents like toluene that are found in paint thinners and adhesives. Because of their widespread commercial use and availability, inhalants are often among the first drugs that children encounter and use of these compounds is often associated with adverse acute and long-term consequences. The cellular and molecular sites of action for abused inhalants is not well known although recent studies report that toluene and other organic solvents alter the activity of specific ligand- and voltage-gated ion channels that regulate cellular excitability. As part of an ongoing effort to define molecular sites of action for abused inhalants, this study examined the effect of toluene on the function of gap junction proteins endogenously expressed in human embryonic kidney (HEK 293) cells. Gap junctions allow cell-to-cell electrical communication as well as passage of small molecular weight substances and are critical for synchronizing cellular activity in certain tissues. Gap junction currents in HEK 293 cells were measured during brief voltage steps using patch-clamp electrophysiology and were blocked by known gap junction blockers confirming expression of connexin proteins in these cells. Toluene dose-dependently inhibited these conductances with threshold effects appearing at approximately 0.4 mM and near complete inhibition occurring at concentrations of 1 mM and higher. The estimated EC50 value for toluene inhibition of gap junction currents in HEK 293 cells was 0.57 mM. The results of these studies suggest that volatile solvents including toluene may produce some of their effects by disrupting inter-cellular communication mediated by gap junction proteins.

Estrogen attenuates glutamate-induced cell death by inhibiting Ca2+ influx through L-type voltage-gated Ca2+ channels.

Estrogen-mediated neuroprotection is observed in neurodegenerative disease and neurotrauma models; however, determining a mechanism for these effects has been difficult. We propose that estrogen may limit cell death in the nervous system tissue by inhibiting increases in intracellular free Ca(2+). Here, we present data using VSC 4.1 cell line, a ventral spinal motoneuron and neuroblastoma hybrid cell line. Treatment with 1 mM glutamate for 24 h induced apoptosis. When cells were pre-treated with 100 nM 17beta-estradiol (estrogen) for 1 h and then co-treated with glutamate, apoptotic death was significantly attenuated. Estrogen also prevented glutamate-mediated changes in resting membrane potential and membrane capacitance. Treatment with either 17 alpha-estradiol or cell impermeable estrogen did not mimic the findings seen with estrogen. Glutamate treatment significantly increased both intracellular free Ca(2+) and the activities of downstream proteases such as calpain and caspase-3. Estrogen attenuated both the increases in intracellular free Ca(2+) and protease activities. In order to determine the pathway responsible for estrogen-mediated inhibition of these increases in intracellular free Ca(2+), cells were treated with several Ca(2+) entry inhibitors, but only the L-type Ca(2+) channel blocker nifedipine demonstrated cytoprotective effects comparable to estrogen. To expand these findings, cells were treated with the L-type Ca(2+) channel agonist FPL 64176, which increased both cell death and intracellular free Ca(2+), and estrogen inhibited both effects. From these observations, we conclude that estrogen limits glutamate-induced cell death in VSC 4.1 cells through effects on L-type Ca(2+) channels, inhibiting Ca(2+) influx as well as activation of the pro-apoptotic proteases calpain and caspase-3.

Calpain activation in apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons exposed to glutamate: calpain inhibition provides functional neuroprotection.

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.