A neurosteroid analogue with T-type calcium channel blocking properties is an effective hypnotic, but is not harmful to neonatal rat brain.

BACKGROUND: More than 4 million children are exposed annually to sedatives and general anaesthetics (GAs) in the USA alone. Recent data suggest that common GAs can be detrimental to brain development causing neurodegeneration and long-term cognitive impairments. Challenged by a recent US Food and Drug Administration (FDA) warning about potentially neurotoxic effects of GAs in children, there is an urgent need to develop safer GAs. METHODS: Postnatal Day 7 (P7) rat pups of both sexes were exposed to six (repeated every 2 h) injections of equipotent hypnotic doses of ketamine or the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) for 12 h. Loss of righting reflex was used to assess hypnotic properties and therapeutic index; quantitative caspase-3 immunohistochemistry was used to assess developmental neuroapoptosis; patch-clamp recordings in acute brain slices were used to assess the effects of 3ß-OH on neuronal excitability and synaptic transmission. Cognitive abilities of rats exposed to ketamine, 3ß-OH, or vehicle at P7 were assessed in young adulthood using the radial arm maze. RESULTS: The neuroactive steroid 3ß-OH has a therapeutic index similar to ketamine, a commonly used clinical GA. We report that 3ß-OH is safe and, unlike ketamine, does not cause neuroapoptosis or impair cognitive development when administered to P7 rat pups. Interestingly, 3ß-OH blocks T-type calcium channels and presynaptically dampens synaptic transmission at hypnotically-relevant brain concentrations, but it lacks a direct effect on γ-aminobutyric acid A or glutamate-gated ion channels. CONCLUSIONS: The neurosteroid 3ß-OH is a relatively safe hypnotic that warrants further consideration for paediatric anaesthesia.

In vitro evaluation of surface properties of Pro Seal® and Opal® SealTM in preventing white spot lesions.

OBJECTIVES: To evaluate the surface properties of two commercially available sealants (Pro Seal® (PS) and Opal® SealTM (OS)) in terms of fluoride(F) release, biofilm formation of Streptococcus mutans and Lactobacillus and the ability to resist acid penetration. SETTING: University of Nebraska Medical Center. MATERIAL & METHODS: Discs of similar diameter and thickness were made from OS and PS. Discs were soaked in double-distilled water, and F released was measured with fluoride meter daily for 14 consecutive days, then at 21 and 28 days. Biofilm formation was evaluated with Streptococcus mutans and Lactobacilli grown on sealant discs using confocal microscopy. Extracted human teeth (n=8) with sealant-coated buccal surfaces and untreated lingual surfaces were exposed to 0.1M lactic acid(pH=4.5) to test the acid penetration. After 1-4 weeks of exposure, teeth were subjected to microhardness testing and SEM microscopy. RESULTS: PS released significantly higher levels of F than OS. PS showed more S. mutans adherence than OS, whereas Lactobacillus did not show any differences in adherence. Both sealants protected enamel surfaces, showing statistically significant difference in the depth of acid penetration compared to their unsealed control sides. CONCLUSION: F release was adequate to aid in remineralization, although clinically it would not likely aid in preventing demineralization as there was no prolonged release of F by both sealants tested. S. mutans adherence to OS surface was less compared to PS surface, which could be of relevance in biofilm formation and white spot lesions. Both sealants protected enamel surfaces from acid penetration.

Conversion From Limb Salvage to Late Amputation: Lessons Learned From Recent Battlefields With Application to Civilian Trauma.

Battlefield injuries and high-energy civilian trauma present orthopaedic surgeons with treatment challenges. Despite efforts at limb salvage, some patients elect late amputation. This article reviews risk factors that predispose to late amputation. Using a MEDLINE search, English language peer-reviewed articles from 1993 to 2013 having data on late amputation following limb salvage were included. Late lower extremity amputation after limb salvage varied from 3.9% to 40% in civilian patients and from 5.2% to 15.2% in military patients. Factors influencing a patient's decision to undergo late amputation included a combination of complex pain symptoms with neurologic dysfunction, infection, a desire for improved limb functionality, and unwillingness to endure an often complicated and lengthy course of treatment. In military patients, rank was a significant risk factor since officers were 2.5 times more likely to elect late amputation (p < .05) than enlisted personnel. Despite often extraordinary efforts towardlimb salvage, results maybe disappointing.

Field tourniquets in an austere military environment: A prospective case series.

OBJECTIVE: Field tourniquets are often used for battlefield extremity injuries. Their effectiveness has been documented by a large combat theater trauma center. However, their use and effectiveness by an austere forward surgical team has not been reported. Aims of this study were to determine: Whether field tourniquets: (1) Were placed for appropriate indications; (2) significantly reduced hemorrhage as measured by transfusion requirements; (3) influenced vital signs and injury severity scores; and (4) did they cause limb amputation, changed amputation level, or other complications. METHODS: Twenty-five patients with 30 involved extremities presenting to a forward surgical team in Iraq met the inclusion criteria. We prospectively collected data regarding the presence, indications for, and effectiveness of field tourniquets based on the need for blood transfusion. We recorded any complications associated with their use. RESULTS: Tourniquets significantly reduced hemorrhage from penetrating injuries as measured by transfusion requirements. Those having major vascular injuries with effective tourniquets, a total of 12 units of blood were transfused (1.7 units/vascular injury; 2 units/patient). However, 19 units were transfused in patients (3.3 units/vascular injury; 3.8 units/patient) who had an ineffective or no tourniquet (p = 0.0006). Transfusion requirements were related the presence of an effective tourniquet regardless of concomitant injuries. The group with effective tourniquets and compressed hemorrhage presented with higher mean systolic (p = 0.003) and diastolic (p = 0.023) blood pressures than the group with no tourniquets or ineffective ones. Complications included one peroneal nerve palsy and no amputations resulted from tourniquet application. CONCLUSION: Field tourniquets applied for penetrating injuries with severe bleeding can significantly reduce transfusion requirements and help maintain adequate blood pressure. Tourniquets were not the proximate cause of amputation and did not determine the choice of immediate amputation level.

Alpha-substituted gamma-butyrolactones: new class of anticonvulsant drugs.

Alkyl-Substituted gamma-butyrolactones were synthesized and tested for their convulsant and anticonvulsant actions in mice and guinea pigs. The alpha-substituted compounds, alpha, alpha-dimethyl-, and alpha-ethyl-alpha-methyl-gamma-butyrolactone were anticonvulsant compounds with a spectrum of activity similar to that of ethosuximide. In contrast, beta-substituted compounds were convulsant agents similar to picrotoxinin. The alpha-substituted-gama-butyrolactones represent a new class of anticonvulsant drug with experimental and clinical potential.

Subacute High-Energy Traumatic Knee Dislocation in an Ambulatory Patient: A Case Report.

CASE: A 62-year-old man presented with a posterolateral knee dislocation sustained in a motorcycle collision initially managed nonsurgically. His treatment was complicated by recurrent dislocation from noncompliance with bracing and weight-bearing restrictions and by comorbidities including peripheral artery disease, type 2 diabetes, and heavy smoking and drinking. He was treated with a multiplanar knee-spanning external fixator and patellar tendon repair without further ligamentous reconstruction. This treatment provided a stable, functional knee with minimal pain. CONCLUSIONS: In this complex, noncompliant patient with a subacute knee dislocation, knee-spanning biplanar external fixation and patellar tendon repair without ligament reconstruction were crucial in achieving a satisfactory outcome.

Developmental alteration in GABAA receptor structure and physiological properties in cultured cerebellar granule neurons.

Although we now have extensive knowledge about the GABAA receptor subunits determining benzodiazepine modulation of channel function, little is known about subunits influencing other modulatory sites on the GABAA receptor-chloride channel complex. We have identified a developmental change in subunit composition of the GABAA receptor in cultured cerebellar granule neurons that eliminates benzodiazepine-mediated enhancement of GABA responses and alters modulation by a substituted gamma-butyrolactone. Based on data from sequential PCR experiments, we mimicked the functional properties of early and mature receptors with heterologous expression of specific subunit combinations. This report describes one of the most extensive cell- and site-specific developmental changes for an ion channel seen to date.

Cyclodextrins sequester neuroactive steroids and differentiate mechanisms that rate limit steroid actions.

BACKGROUND AND PURPOSE: Neuroactive steroids are potent modulators of GABA(A) receptors and are thus of interest for their sedative, anxiolytic, anticonvulsant and anaesthetic properties. Cyclodextrins may be useful tools to manipulate neuroactive effects of steroids on GABA(A) receptors because cyclodextrins form inclusion complexes with at least some steroids that are active at the GABA(A) receptor, such as (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha5alphaP, allopregnanolone). EXPERIMENTAL APPROACH: To assess the versatility of cyclodextrins as steroid modulators, we investigated interactions between gamma-cyclodextrin and neuroactive steroids of different structural classes. KEY RESULTS: Both a bioassay based on electrophysiological assessment of GABA(A) receptor function and optical measurements of cellular accumulation of a fluorescent steroid analogue suggest that gamma-cyclodextrin sequesters steroids rather than directly influencing GABA(A) receptor function. Neither a 5beta-reduced A/B ring fusion nor a sulphate group at carbon 3 affected the presumed inclusion complex formation between steroid and gamma-cyclodextrin. Apparent dissociation constants for interactions between natural steroids and gamma-cyclodexrin ranged from 10-60 microM. Although gamma-cyclodextrin accommodates a range of natural and synthetic steroids, C(11) substitutions reduced inclusion complex formation. Using gamma-cyclodextrin to remove steroid not directly bound to GABA(A) receptors, we found that cellular retention of receptor-unbound steroid rate limits potentiation by 3alpha- hydroxysteroids but not inhibition by sulphated steroids. CONCLUSIONS AND IMPLICATIONS: We conclude that gamma-cyclodextrins can be useful, albeit non-specific, tools for terminating the actions of multiple classes of naturally occurring neuroactive steroids.

A new hypothesis based on suicide substrate inhibitor studies for the mechanism of action of aromatase.

Recently, it was discovered that 4-hydroxy-4-androstene-3,17-dione, 4-androstene-3,6,17-trione, and 1,4,6-androstatriene-3,17-dione, compounds previously reported to be competitive inhibitors of aromatase, cause a time-dependent loss of aromatase activity in human placental microsomes. We report here that 1,4-androstadiene 3,17-dione (Ki 0.32 microM; kinact 0.91 X 10(-3)/sec) and testolactone (Ki 35 microM; kinact 0.36 X 10(-3)/sec) also cause a similar loss of aromatase activity. The mechanism which explains the unexpected loss of activity caused by these five inhibitors is neither established nor apparent from current theories of the enzyme mechanism of action of aromatase. We propose an inactivation mechanism based on a new hypothesis for estrogen biosynthesis in which the third enzyme oxidation carried out by aromatase results in the formation of an enzyme-bound intermediate. This intermediate is released as an aromatized product via a facile elimination reaction which simultaneously regenerates the unaltered active enzyme. Various structural modifications made in these five inhibitors are hypothesized to redirect this elimination reaction so that the steroid intermediate remains covalently attached to the enzyme instead of being released as an aromatized product.

Differential effect of alpha-difluoromethylornithine on the in vivo uptake of 14C-labeled polyamines and methylglyoxal bis(guanylhydrazone) by a rat prostate-derived tumor.

The uptake of exogenously administered radiolabeled polyamines by a rat prostate-derived tumor line, the Dunning R3327 MAT-Lu, and various normal tissues was studied. Pretreatment of tumor cells in vitro with alpha-difluoromethylornithine (DFMO), a polyamine synthesis inhibitor, resulted in a markedly enhanced uptake of both [14C]putrescine and [14 C]spermidine. The in vitro uptake of [14C]putrescine by these cells was effectively inhibited by unlabeled spermine, spermidine, 1,8-diaminooctane, 1,7-diaminoheptane, 1,6-diaminohexane, 1,5-diaminopentane, 1,4-diaminopentane, and 1,4-diaminobutane, but less effectively by 1,4-diamino-2,3-butene and 1,4-diamino-2,3-butyne. The diamines, 1,3-diaminopropane and 1,2-diaminoethane, were ineffective in inhibiting [14C]putrescine uptake in vitro into the R3327 MAT-Lu cell line. When tumor-bearing animals were pretreated with DFMO or with DFMO and 5-alpha-dihydrotestosterone propionate, the tumor and prostate uptake of [14C]putrescine and [14C]-cadaverine was enhanced but not substantially increased in other tissues. In contrast to the in vitro results, spermidine and spermine were not enhanced substantially by DFMO pretreatment into any tissue, and their uptake into the tumor actually decreased. Ethylenediamine, which does not utilize the polyamine transport system, did not have its uptake increased into any tissue following DFMO pretreatment. The chemotherapeutic agent, methylglyoxal bis(guanylhydrazone), which utilizes the polyamine transport system for uptake into cells, exhibited uptake behavior different from that of the polyamines. Thus, methylglyoxal bis(guanylhydrazone) uptake into the tumor was not significantly increased or decreased by DFMO or by DFMO + 5-alpha-dihydrotestosterone propionate pretreatment, and only the ventral, but not the dorsal-lateral, lobe of the prostate showed increased uptake of methylglyoxal bis(guanylhydrazone) following DFMO + 5-alpha-dihydrotestosterone propionate pretreatment.