Alkalinization and precipitation characteristics of 0.2% ropivacaine.

BACKGROUND AND OBJECTIVES: Alkalinization of local anesthetics has been used to increase the speed of onset of nerve blocks. However, alkalinization of local anesthetic solutions may cause precipitation, thereby decreasing bioavailability and anesthetic activity. Alkalinization of ropivacaine has not been described. This laboratory study assessed the alkalinization and precipitation characteristics of ropivacaine. METHODS: Aliquots (2 mL) of commercially available ropivacaine (Naropin, 0.2%; Astra Pharmaceutical, Westborough, MA) were alkalinized with increasing amounts (0.01, 0.02, 0.04 mL) of sodium bicarbonate (8.4%) and immediately monitored for pH change and onset of visible precipitation at room temperature. We then alkalinized ropivacaine with sodium bicarbonate and measured the amount of precipitate that accumulated after various incubation times. RESULTS: The pH of ropivacaine increases with the addition of small amounts of bicarbonate. The calculated percentage of nonionized ropivacaine increased from 0.3% to greater than 30% with alkalinization from pH of 5.51 to 7.63. Drug loss to precipitation increased with higher doses of bicarbonate, reaching 25% to 30% of the total ropivacaine. Even with a low dose of bicarbonate (0.1 mL bicarbonate/20 mL ropivacaine), precipitation increased with time of incubation, reaching a plateau at 20 minutes. CONCLUSIONS: A laboratory evaluation that establishes the alkalinization characteristics of ropivacaine is a prerequisite for designing a clinical study of alkalinized ropivacaine. In our experiment, low doses of bicarbonate produced significant increases in the proportion of nonionized ropivacaine with only modest precipitation. There would be a low likelihood of substantial drug precipitation if the mixture was administered within 5 to 10 minutes after alkalinization. These results indicate that alkalinized ropivacaine should not be used for infusions and that ropivacaine should not be alkalinized until just before use.

Activation of adenosine A1 and A2A receptors modulates dopamine D2 receptor-induced responses in stably transfected human neuroblastoma cells.

Adenosine can influence dopaminergic neurotransmission in the basal ganglia via postsynaptic interaction between adenosine A2A and dopamine D2 receptors. We have used a human neuroblastoma cell line (SH-SY5Y) that was found to express constitutively moderate levels of adenosine A1 and A2A receptors (approximately 100 fmol/mg of protein) to investigate the interactions of A2A/D2 receptors, at a cellular level. After transfection with human D2L receptor cDNA, SH-SY5Y cells expressed between 500 and 1,100 fmol of D2 receptors/mg of protein. In membrane preparations, stimulation of adenosine A2A receptors decreased the affinity of dopamine D2 receptors for dopamine. In intact cells, the calcium concentration elevation induced by KCI treatment was moderate, and dopamine had no effect on either resting intracellular free Ca2+ concentration ([Ca2+]i) or KCI-induced responses. In contrast, pretreatment with adenosine deaminase for 2 days dramatically increased the elevation of [Ca2+]i evoked by KCI, which then was totally reversed by dopamine. The effects induced by 48-h adenosine inactivation were mimicked by application of adenosine A1 antagonists and could not be further reversed by acute activation of either A1 or A2A receptors. Acute application of the selective A2 receptor agonist CGS-21680 counteracted the D2 receptor-induced [Ca2+]i responses. The present study shows that SH-SY5Y cells are endowed with functional adenosine A2A and A1 receptors and that A2A receptors exert an antagonistic acute effect on dopamine D2 receptor-mediated functions. In contrast, A1 receptors induce a tonic modulatory role on these dopamine functions.

Management of multiple intracranial aneurysms: neuroanesthetic considerations of sickle cell disease.

Intracranial aneurysms are a common complication of sickle cell disease. The management of a patient with multiple intracranial aneurysms and sickle cell disease is described. The English language literature is reviewed. Neuroanesthetic management has traditionally been based on the avoidance of factors said to lead to erythrocyte sickling; however neuropathology typically arises from arterial intimal damage, not from venous sickling. Neuroanesthesia should be based on an appreciation of this pathophysiological model. Consideration of precipitants of vaso-occlusive crises, such as hypothermia, dehydration and possibly altered hemodynamics, should influence management.

Variations in the composition of spinal anesthetic solutions: the effects of drug addition order and preparation methods.

UNLABELLED: Adjuvants such as opioids or epinephrine are commonly added in small volumes to multicomponent spinal anesthetic solutions. In this study, we tested the hypothesis that final adjuvant concentrations vary depending on the devices and techniques used to prepare the anesthetic solution. We compared two aspiration devices, the filter needle and the filter straw, in a laboratory study. Two techniques for drawing up and estimating adjuvant volumes were assessed, as was variation in the composition of a model spinal anesthetic solution resulting from intra- and interindividual variability. A model hyperbaric anesthetic solution consisting of tetracaine, dextrose, and methylene blue (MB) as a small-volume tracer solution was studied. The components were drawn up into a syringe through one of two commercially supplied aspiration devices, a filter straw or a filter needle. The effect of the order of aspiration of the components into the syringe was measured by determining the MB concentration in the final solution by optical absorbance. Ten experienced anesthesiologists then prepared samples of the test solution using one of two different techniques to estimate tracer volume in the aspiration syringe. In comparison studies, the MB tracer was added to the hyperbaric solution with a tuberculin syringe. The order of aspiration of the solution components had a large effect on the final concentration of the MB tracer in the ultimate mixture. Variation in the MB concentration was on the order of four- to fivefold. Effects were larger for the filter straw compared with the filter needle. A comparison of 10 anesthesiologists revealed large intra- and interindividual variations in the final composition of the model anesthetic solution. The concentration of tracer added to the mixture with a tuberculin syringe approximated the planned yield. We conclude that the devices and techniques used to prepare mixtures of drugs for delivery to the cerebrospinal fluid may influence the concentrations of drugs in the anesthetic and, thus, the dose supplied to the patient receiving spinal anesthesia. Variation in clinical effects of spinal anesthetics may be attributable, in part, to variation in the composition of the anesthetic. IMPLICATIONS: This laboratory study demonstrates the potential for large variation in the composition of spinal anesthetic mixtures.

Agonist-induced desensitization of A2B adenosine receptors.

Agonist-induced desensitization has been described for the A1, A2A, and A3 adenosine receptor subtypes of the G protein-coupled receptor superfamily. Desensitization of the fourth adenosine receptor subtype, the A2B adenosine receptor (A(2B)R), has not been studied extensively. We sought to determine whether the A(2B)R is subject to agonist-induced desensitization. COS 7 cells, which exhibit endogenous expression of the A(2B)R, and transfected CHO cells, which stably express a modified rat A(2B)R bearing a 5' FLAG epitope tag, were studied. Cyclic AMP (cAMP) responsiveness to an acute challenge was measured after pretreating (desensitizing) cells with the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). Incubation with NECA resulted in hyporesponsiveness to acute agonist challenge in both COS 7 and transfected CHO cells. Desensitized cells exhibited restoration of cAMP responses after recovery for 24 hr in growth medium. Choleratoxin-induced cAMP responses were preserved in desensitized cells, and high concentrations of NECA were unable to overcome the desensitization. Membrane levels of the epitope-tagged A(2B)R were assessed by western blot in transiently transfected COS 7 cells. The expression of epitope-tagged A(2B)Rs was not different between control and desensitized cells. In northern blot analysis, levels of endogenous A(2B)R mRNA were similar in control and desensitized COS 7 cells. We conclude that the A(2B)R is subject to agonist-induced desensitization with preserved expression of A(2B)R mRNA and protein. Uncoupling of the A2B adenosine receptor from the G protein complex may contribute to the mechanism of desensitization.

Regulation of mu opioid receptor mRNA levels by activation of protein kinase C in human SH-SY5Y neuroblastoma cells.

BACKGROUND: The mu opioid receptor (MuOR) is a member of the superfamily of G protein-coupled receptors that mediates the analgesic actions of endogenous opioid peptides and the narcotic alkaloid derivatives of morphine. Activation and translocation of protein kinase C (PKC) by N-methyl-D-aspartate receptor stimulation correlates with resistance to opioid drugs in experimental states of neuropathic pain, but the cellular mechanisms of resistance have not been identified. One possibility is that PKC activation regulates MuOR mRNA expression and thus the ability to generate functional receptors. Using a human neuroblastoma cell line, the authors tested the hypothesis that phorbol ester activation of PKC regulates MuOR mRNA levels. METHODS: SH-SY5Y cells were maintained in a continuous monolayer culture and treated with phorbol esters or other agents before extraction of total cellular RNA. Slot-blot hybridization was used to measure the level of MuOR mRNA using 32P-labeled MuOR cDNA probes under high-stringency conditions. Autoradiograms were analyzed by scanning and densitometry. RESULTS: MuOR mRNA levels decreased in a dose- and time-dependent manner after tetradecanoyl phorbol acetate (TPA) was administered to activate PKC. The nadir, a level of approximately 50% of control, was at 2-8 h, followed by gradual recovery. The actions of TPA were blocked by pretreatment with the selective PKC inhibitor bisindolylmaleimide, but not by inhibition of protein synthesis with cycloheximide or anisomycin. The combination of TPA treatment and transcription inhibition with actinomycin D was associated with a transient increase in MuOR mRNA. CONCLUSIONS: Mu opioid receptor mRNA levels are regulated by activation of PKC in a neuronal model. Protein kinase C effects which decrease MuOR mRNA levels appear largely independent of new protein synthesis, and cytotoxicity does not account for the findings. Plasticity of MuOR gene expression may contribute to variations in clinical responses to opioid analgesics in clinical states such as neuropathic pain.

Compartment syndrome of the upper arm after pressurized infiltration of intravenous fluid.

Perioperative management of the acute trauma patient requires constant surveillance for the unexpected. We describe a case of upper extremity compartment syndrome resulting from pressurized infusion of crystalloid through an intravenous catheter placed in the emergency ward. Early recognition, diagnosis, and intervention with surgical fasciotomy averted potential complications and morbidity.

Protein kinase C regulates adenosine A2a receptor mRNA expression in SH-SY5Y cells.

Protein kinase C regulates mRNAs encoding several G protein-linked receptors but its role in adenosine A2a receptor expression is not known. We tested the hypothesis that protein kinase C activated by tetradecanoyl phorbol acetate (TPA) regulates adenosine A2a receptor mRNA levels. SH-SY5Y human neuroblastoma cells express adenosine receptors which positively couple to adenylyl cyclase with a pharmacologic profile expected of the A2a subtype. Northern blotting demonstrated an adenosine A2a receptor mRNA species of similar molecular size in SH-SY5Y cells and in human brain. TPA increased adenosine A2a receptor mRNA in a dose- and time-dependent fashion. Transcription or translation inhibition prevented increases in adenosine A2a receptor mRNA. Bisindolylmaleimide blocked TPA effects. Adenosine A2a receptor mRNA stability was unchanged by TPA. This study identifies a human neuroblastoma cell line expressing functional adenosine A2a receptors. Protein kinase C activation appears to enhance transcription of the adenosine A2a receptor gene.

Transient compartment syndrome of the forearm after attempted radial artery cannulation.

Radial artery cannulation for continuous intraoperative monitoring of arterial blood pressure is considered a safe procedure. One complication of arterial cannulation is hematoma formation at the time of insertion or removal of the catheter. Bleeding is usually self-limited or will stop with compression without significant sequelae, even in the anticoagulated patient. We describe a case of hematoma with a transient compartment syndrome of the forearm after attempts to cannulate the radial artery for intraoperative monitoring purposes.

Characterization and expression of the human A2a adenosine receptor gene.

The actions of the neurotransmitter adenosine are mediated by a family of high-affinity, G protein-coupled receptors. We have characterized the gene for the human A2a subtype of adenosine receptor (hA2aR) and determined levels of A2aR mRNA in human brain regions and nonneural tissues. Human genomic Southern blot analysis demonstrates the presence of a single gene encoding the hA2aR located on chromosome 22. Two overlapping cosmids containing the hA2aR gene were isolated from a chromosome 22 library and characterized. Southern blot and sequence analyses demonstrate that the hA2aR gene spans approximately 9-10 kb with a single intron interrupting the coding sequence between the regions encoding transmembrane domains III and IV. The sequence of the hA2aR gene diverged from the reported cDNA structure in the 5' untranslated region. This divergence appears to result from an artifact in the construction of the original cDNA library. By northern blot analysis, high expression of the hA2aR gene was identified in the caudate nucleus with low levels of expression in other brain regions. High expression was also seen in immune tissues; lesser A2aR expression was detected in heart and lung. The gene for the A2a subtype of receptor for the neurotransmitter adenosine falls in the class of intron containing G protein-coupled receptor genes. Expression in the basal ganglia is consistent with a role for the hA2aR in motor control. Activation of the A2aR may also regulate immune responses and cardiopulmonary function.

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion.

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.

The delivery of aerosolized steroids from MDIs with nozzle extensions: quantitative laboratory evaluation of a method to improve aerosol delivery to intubated patients.

OBJECTIVE: Pulmonary deposition of aerosolized drug from a metered dose inhaler (MDI) is low with intubated patients. In the laboratory, extension of the MDI nozzle to the endotracheal tube tip has been shown to increase the delivered dose of albuterol. The objectives of this study were to determine the dose of aerosolized steroid (beclomethasone and triamcinolone) delivered through a MDI nozzle extension, the effect of nozzle extension length and number of actuations on the delivered dose, and particle size delivered through the nozzle extension. DESIGN: A 19-G catheter was used as the MDI nozzle extension. The nozzle extension was attached to a 60-ml syringe via the Luer-Lok connection, and the distal end was directed through a hole drilled into a 15-ml capped tube. The MDI was placed into the syringe and actuated by pressing the syringe plunger. Drug delivered through the nozzle extension into the tube was dissolved in methanol (beclomethasone) or ethanol (triamcinolone). Nozzle extension lengths of 10 cm, 20 cm and 30 cm were studied. For each nozzle extension length, delivery was assessed using one, two, three and five actuations of each drug. Drug remaining in the nozzle extension was recovered by rinsing with the appropriate solvent. Aerosol particle size leaving the nozzle extension was determined using a seven-stage cascade impactor. Beclomethasone and triamcinolone concentrations were determined by spectrophotometry at 239 nm. SETTING: Respiratory care laboratory of a university teaching hospital. RESULTS: For the pooled results, 70.2 +/- 14.1% of the dose was delivered through the nozzle extension, with no difference between beclomethasone and triamcinolone (p = 0.838). The proportion of drug delivered through the 10-cm extension (76.7 +/- 8.4%) was greater than that from the 20-cm (66.1 +/- 16.5%) and 30-cm (67.7 +/- 13.9%) extensions (p = 0.001). Less drug was delivered through the extension with one actuation (54.1 +/- 17.7%) than with two (71.2 +/- 7.7%), three (77.2 +/- 5.5%), or five actuations (78.2 +/- 4.3%) (p < 0.001). There was a decrease in MMAD with increasing nozzle extension length (3.14 +/- 0.61 microns for 10 cm, 2.97 +/- 0.28 microns for 20 cm, 2.37 +/- 0.27 microns for 30 cm; p = 0.005). CONCLUSIONS: A high proportion of aerosolized steroid was delivered with a MDI actuated through a nozzle extension. The proportion delivered through the nozzle extension was significantly less with longer nozzle extensions and with fewer actuations, but this may not be clinically important. Although particle sizes were smaller from longer nozzle extensions, all were within the respirable range. These results suggest that steroids can be delivered efficiently using a MDI nozzle extension.

Syringe-actuated metered dose inhalers: a quantitative laboratory evaluation of albuterol delivery through nozzle extensions.

The metered dose inhaler (MDI) is a device that can be used to supply aerosolized bronchodilators to the tracheobronchial tree of patients requiring endotracheal intubation. Because direct actuation of the MDI into the breathing circuit is inefficient, a technique of extending the MDI nozzle with small-bore, long, intravenous catheters has been devised. To facilitate connecting the MDI to the extension and actuating the release of aerosol, insertion of the MDI cannister into the barrel of a large syringe attached to the Luer hub of the extension has been proposed but not quantitatively evaluated. The purpose of this study was to test, in a laboratory model, the performance of syringe-actuated MDIs attached to nozzle extensions. The deposition of the bronchodilator albuterol in the delivery device and distal to the extension was quantified spectrophotometrically. Maximum distal delivery of drug from nozzle extensions fashioned from small-bore (19-gauge) catheters was 80% of the actuated release. Loss of drug to the inner surface of the syringe plus extension varied inversely with the number of successive actuations. Syringes from two manufacturers in two sizes were compared and found to perform comparably. Syringes could be attached to the Luer connector of the secondary sampling channel of special pediatric-sized endotracheal tubes (ETTs) and the channels were evaluated as nozzle extensions, with distal delivery > 80% as efficient as the 19-gauge intravenous catheter. We conclude that syringe actuation of MDIs through nozzle extensions is an efficient method for supplying the aerosol distal to the tip of the ETT.(ABSTRACT TRUNCATED AT 250 WORDS)

Small bore nozzle extensions to improve the delivery efficiency of drugs from metered dose inhalers: laboratory evaluation.

Metered dose inhalers (MDIs) are frequently used to supply aerosolized drugs, particularly bronchodilators, to the tracheobronchial tree of patients with endotracheal tubes in the intensive care unit or in the operating room. The efficiency of delivery to the lungs of agents such as the beta 2-adrenergic agonists is known to be low. In an in vitro model, we evaluated a means of improving the delivery of drug released from an MDI beyond the distal tip of the endotracheal tube. Extensions of the MDI nozzle were fashioned from modified intravenous catheters or sections of small bore polyethylene tubing. A model trachea/carina was constructed and suspended above a collecting device. An albuterol MDI was actuated through the nozzle extension and into the model airway. We measured the quantity of albuterol deposited in the nozzle extension, in the trachea/carina and in the distal collecting device. Particle size distribution was determined with a cascade impactor. The results indicate an inverse relationship between the quantity of drug delivered distally and the inner diameter of the nozzle extension, with a marked increase in delivery for an inner diameter < 1 mm. Ninety percent of the actuated dose from the MDI exited a 0.76-mm inner diameter nozzle extension. From 20 to 30% of the nominal MDI dose was recovered from the distal collector, 70% of which deposited in the particle size range of 1 to 5 microns. Deposition in the trachea/carina was high, but this was reduced by introducing a flare in the tip of the nozzle extension, which did not affect the dose reaching the distal collector.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum.

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

pH adjustment of local anesthetic solutions with sodium bicarbonate: laboratory evaluation of alkalinization and precipitation.

pH adjustment of several commonly administered local anesthetic solutions was evaluated in the laboratory. The pH achieved after the addition of sodium bicarbonate and the onset of precipitation for alkalinized solutions were recorded. Solutions of 2-chloroprocaine and lidocaine readily alkalinized to near physiological pH without precipitation. Mepivacaine solutions exhibited a tendency for delayed precipitation (18-20-minute latency for 1.5% mepivacaine) above neutral pH. Bupivacaine and etidocaine solutions precipitated after the addition of small amounts of sodium bicarbonate and could not be alkalinized to physiologic pH. Two commercially available sodium bicarbonate preparations, 4% (wt/vol) and 8.4% (wt/vol), were compared and produced similar pH changes and precipitation behavior. The data obtained for pH and time to precipitation for local anesthetic solutions alkalinized with sodium bicarbonate may be useful for practical application in the clinical setting.

Local anesthetics inhibit veratridine-induced secretion of calcitonin gene-related peptide (CGRP) from cultured rat trigeminal ganglion cells.

Secretion of calcitonin gene-related peptide (CGRP) was studied with the model system of dispersed adult rat trigeminal ganglion cells. Veratridine stimulated secretion of CGRP immunoreactivity. Tetrodotoxin and local anesthetics inhibited veratridine-stimulated peptide secretion. These observations implicate sodium channels in CGRP secretion and are consistent with a role for the peptide as an extracellular neuromodulator in the sensory nervous system.

Thyroid hormones reversibly suppress somatostatin secretion and immunoreactivity in cultured neocortical cells.

Thyroid hormone effects on brain somatostatin-like immunoreactivity (SRIF-LI) were studied in an in vitro model system. Serum was removed from the nutrient culture medium of fetal day-18 rat cerebral cortex cells maintained in primary, long-term, dispersed monolayer culture. Chronic administration of either T3 or T4 in serum-free medium was associated with suppressed release of SRIF-LI into the culture medium (36-43 h accumulation), cell content of peptide and acute release in response to potassium-induced depolarization. Suppression was dose-dependent with an IC50 of less than 1 nM for T3. The most dramatic effects were observed for K+-induced release. Thirty-five to 50% suppression was typically observed with T3 at a near maximum dose (3 nM). Reverse T3 and diiodotyrosine were less potent and effective than T3. TRIAC and diiodothyronine also possessed significant suppressive activity. T3 suppression of release depended on duration of pretreatment. Administered for less than 16 h, T3 failed to significantly suppress K+-induced release, but significant suppression was observed for pretreatment periods of 16 h or longer. Indirect fluorescent immunohistochemical examination revealed a reduction in the number of cells positively stained for SRIF-LI in T3-treated dishes relative to controls. Upon removal of T3 and subsequent recovery in serum supplemented medium for 24 h, T3-treated and control cells exhibited similar levels of SRIF-LI release and cell content. T3-treated and control cells incorporated [3H] leucine into trichloracetic acid precipitable counts to similar extents. Dexamethasone and several sex steroids failed to modify the effects of T3 and did not independently influence SRIF-LI levels. Acute cycloheximide administration did not reverse T3 effects. The data indicate that primary brain cell cultures may be useful models to examine direct peripheral hormone actions on nervous tissue. Thyroid hormones suppress SRIF-LI levels in a dose, time and structure-dependent manner, which appears to be reversible. The findings are consistent with a possible integration of peripheral hormone and brain peptide physiology.