Robert J. Brosnan

Analgesia mediated by soluble epoxide hydrolase inhibitors is dependent on cAMP.

Pain is a major health concern even though numerous analgesic agents are available. Side effects and lack of wide-spectrum efficacy of current drugs justify efforts to better understand pain mechanisms. Stabilization of natural epoxy-fatty acids (EFAs) through inhibition of the soluble epoxide hydrolase (sEH) reduces pain. However, in the absence of an underlying painful state, inhibition of sEH is ineffective. Surprisingly, a pain-mediating second messenger, cAMP, interacts with natural EFAs and regulates the analgesic activity of sEH inhibitors. Concurrent inhibition of sEH and phosphodiesterase (PDE) dramatically reduced acute pain in rodents. Our findings demonstrate a mechanism of action of cAMP and EFAs in the pathophysiology of pain. Furthermore, we demonstrate that inhibition of various PDE isozymes, including PDE4, lead to significant increases in EFA levels through a mechanism independent of sEH, suggesting that the efficacy of commercial PDE inhibitors could result in part from increasing EFAs. The cross-talk between the two major pathways—one mediated by cAMP and the other by EFAs—paves the way to new approaches to understand and control pain.

Effects of remifentanil on measures of anesthetic immobility and analgesia in cats

OBJECTIVE To evaluate effects of various doses of remifentanil on measures of analgesia in anesthetized cats. ANIMALS 6 healthy adult cats. PROCEDURES Minimum alveolar concentration (MAC) for isoflurane and thermal threshold responses were evaluated in anesthetized cats. Remifentanil infusions of 0 (baseline), 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, and 16 microg/kg/min were administered; after a 45-minute equilibration period, isoflurane MAC and responses were determined. Isoflurane MAC was determined in anesthetized cats once for each remifentanil infusion rate by use of a standard tail clamp technique. Thermal threshold was measured in awake cats by use of a commercially available analgesiometric probe placed on the lateral portion of the thorax; remifentanil infusions were administered in randomized order to anesthetized cats, and thermal threshold determinations were made by an investigator who was unaware of the infusion rate. RESULTS Mean +/- SEM median effective concentration (EC(50)) for remifentanil and its active metabolite, GR90291, for the thermal threshold test was 1.00 +/- 0.35 ng/mL and 307 +/- 28 ng/mL of blood, respectively. Dysphoria was detected in all awake cats at the 2 highest remifentanil infusion rates. However, isoflurane MAC during remifentanil infusions was unchanged from baseline values, even at blood opioid concentrations approximately 75 times the analgesic EC(50). CONCLUSIONS AND CLINICAL RELEVANCE Immobility and analgesia as reflected by thermal threshold testing were independent anesthetic end points in the cats. Results of MAC-sparing evaluations should not be used to infer analgesic potency without prior validation of an MAC-analgesia relationship for specific drugs and species.

Scintigraphic evaluation of intra‐arterial and intravenous regional limb perfusion of allogeneic bone marrow‐derived mesenchymal stem cells in the normal equine distal limb using 99mTc‐HMPAO

REASONS FOR PERFORMING STUDY Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle-induced iatrogenic damage to the surrounding tissue. OBJECTIVES The purpose of our study was to evaluate MSC distribution after intra-arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. METHODS Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium-99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. RESULTS Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (30-60%) and 28% (14-50%) (median [minimum-maximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. CONCLUSIONS Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. POTENTIAL RELEVANCE Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.

Anesthetic properties of the ketone bodies β-hydroxybutyric acid and acetone

BACKGROUND:We tested the hypothesis that two metabolites that are elevated in ketosis (β-hydroxybutyric acid, and acetone) modulate ion channels in a manner similar to anesthetics and produce anesthesia in animals. METHODS:α1β2&ggr;2s&ggr;-aminobutyric acid type A (GABAA), α1 glycine, NR1/NR2A N-methyl-d-aspartate, and two pore domain TRESK channels were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. The effect of β hydroxybutyric acid and acetone on channel function was measured. The anesthetic effects of these drugs were measured in X. laevis tadpoles. RESULTS:Both β hydroxybutyric acid and acetone enhanced glycine receptor function in the concentration range that is obtained in ketoacidosis in humans. Beta hydroxybutyric acid also enhanced GABAA receptor function at these concentrations. Both acetone and β-hydroxybutyric acid anesthetized tadpoles, with an EC50 for acetone of 264 ± 2 mM (mean ± se) and for β-hydroxybutyric acid of 151 ± 11 mM at pH 7.0. Acetone enhanced GABAA receptors at concentrations of 50 mM and above. Inhibition of TRESK channel function was seen with 100 mM acetone or larger concentration. N-methyl-D-aspartate receptor function was inhibited at concentrations of acetone of 200 mM and larger. CONCLUSIONS:Beta hydroxybutyric acid and acetone are anesthetics. Both ketone bodies enhance inhibitory glycine receptors at concentrations observed clinically in ketoacidosis. In addition, β-hydroxybutyric acid enhances GABAA receptor function at these concentrations. Subanesthetic concentrations of these drugs may contribute to the lethargy and impairment of consciousness seen in ketoacidosis.

Anesthetic Properties of Carbon Dioxide in the Rat

BACKGROUND: Carbon dioxide decreases halothane minimum alveolar concentrations (MAC) in dogs when Paco2 exceeds 95 mm Hg. We sought to confirm these findings for several potent inhaled anesthetics in rats. METHODS: Groups of eight rats were anesthetized with halothane, isoflurane, or desflurane. MAC was determined for each anesthetic alone, and then with increasing concentrations of inspired CO2. A fourth group was given CO2 alone to determine the MAC of CO2. RESULTS: Increasing inspired CO2 concentrations produced a linear dose-dependent decrease in MAC of each potent inhaled anesthetic. With elimination of CO2, the MAC of isoflurane and desflurane returned to the original MAC. As determined by extrapolating these data to 0% of the inhaled anesthetic, the MAC of CO2 was approximately 50% of 1 atm. Given alone, CO2 proved lethal. CONCLUSIONS: Unlike dogs, no threshold for the CO2-MAC response arose with halothane, isoflurane, or desflurane in rats. The ED50 for CO2 is also approximately 50% greater in rats than reported in dogs.

Pharmacokinetics of remifentanil in conscious cats and cats anesthetized with isoflurane

OBJECTIVE To characterize the pharmacokinetics of remifentanil in conscious cats and cats anesthetized with isoflurane. ANIMALS 6 cats. PROCEDURES Remifentanil (1 microg/kg/min for 5 minutes) was administered IV in conscious cats or cats anesthetized with 1.63% isoflurane in oxygen in a randomized crossover design. Blood samples were obtained immediately prior to remifentanil administration and every minute for 10 minutes, every 2 minutes for 10 minutes, and every 5 minutes for 10 minutes after the beginning of the infusion. Blood was immediately transferred to tubes containing citric acid, flash frozen in liquid nitrogen, and stored at -80 degrees C until analysis. Blood remifentanil concentration was determined by use of liquid chromatography-mass spectrometry. Remifentanil concentration-time data were fitted to compartment models. RESULTS A 2-compartment model (with zero-order input because of study design) best described the disposition of remifentanil in awake and isoflurane-anesthetized cats. The apparent volume of distribution of the central compartment, the apparent volume of distribution at steady state, the clearance, and the terminal half-life (median [range]) were 1,596 (1,164 to 2,111) and 567 (278 to 641) mL/kg, 7,632 (2,284 to 76,039) and 1,651 (446 to 29,229) mL/kg, 766 (408 to 1,473) and 371 (197 to 472) mL/min/kg, and 17.4 (5.5 to 920.3) and 15.7 (3.8 to 410.3) minutes in conscious and anesthetized cats, respectively. CONCLUSIONS AND CLINICAL RELEVANCE The disposition of remifentanil in cats was characterized by a high clearance. Isoflurane anesthesia significantly decreased the volume of the central compartment, likely by decreasing blood flow to vessel-rich organs.

Increased NMDA receptor inhibition at an increased Sevoflurane MAC

BackgroundSevoflurane potently enhances glycine receptor currents and more modestly decreases NMDA receptor currents, each of which may contribute to immobility. This modest NMDA receptor antagonism by sevoflurane at a minimum alveolar concentration (MAC) could be reciprocally related to large potentiation of other inhibitory ion channels. If so, then reduced glycine receptor potency should increase NMDA receptor antagonism by sevoflurane at MAC.MethodsIndwelling lumbar subarachnoid catheters were surgically placed in 14 anesthetized rats. Rats were anesthetized with sevoflurane the next day, and a pre-infusion sevoflurane MAC was measured in duplicate using a tail clamp method. Artificial CSF (aCSF) containing either 0 or 4 mg/mL strychnine was then infused intrathecally at 4 μL/min, and the post-infusion baseline sevoflurane MAC was measured. Finally, aCSF containing strychnine (either 0 or 4 mg/mL) plus 0.4 mg/mL dizocilpine (MK-801) was administered intrathecally at 4 μL/min, and the post-dizocilpine sevoflurane MAC was measured.ResultsPre-infusion sevoflurane MAC was 2.26%. Intrathecal aCSF alone did not affect MAC, but intrathecal strychnine significantly increased sevoflurane requirement. Addition of dizocilpine significantly decreased MAC in all rats, but this decrease was two times larger in rats without intrathecal strychnine compared to rats with intrathecal strychnine, a statistically significant (P < 0.005) difference that is consistent with increased NMDA receptor antagonism by sevoflurane in rats receiving strychnine.ConclusionsGlycine receptor antagonism increases NMDA receptor antagonism by sevoflurane at MAC. The magnitude of anesthetic effects on a given ion channel may therefore depend on the magnitude of its effects on other receptors that modulate neuronal excitability.

Sensory Nerve Conduction and Somatosensory Evoked Potentials of the Trigeminal Nerve in Horses with Idiopathic Headshaking

BACKGROUND Idiopathic headshaking (HSK) in horses is a distressing disorder in which the etiology and pathophysiology are unknown. HYPOTHESIS Differences in sensory function of the trigeminal nerve exist between healthy and affected horses. ANIMALS Six healthy mature geldings and 6 mature geldings with idiopathic HSK. METHODS Prospective study. Sensory nerve action and somatosensory evoked potentials studies were performed. The stimulus site comprised the gingival mucosa dorsal to the maxillary canine. A pair of recording electrodes was placed along the sensory pathway of the trigeminal complex at the infraorbital nerve (R1), maxillary nerve (R2), spinal tract of trigeminal (R3), and somatosensory cortex (R4). Sensory nerve action potential latency (ms), amplitude (μV), duration (ms), area under the curve (μVms), and conduction velocity (m/s) were calculated. RESULTS Threshold for activation of the infraorbital branch of the trigeminal nerve was significantly different between 5 affected (≤ 5 mA) and 6 control horses (≥ 10 mA). After initiation of an action potential, there were no differences in all parameters measured and no differences between left and right sides. A horse with seasonal HSK tested during a time of no clinical manifestations showed a threshold for activation similar to control horses. CONCLUSIONS AND CLINICAL IMPORTANCE This study confirms involvement of the trigeminal nerve hyperexcitability in the pathophysiology of disease. Further, results might support a functional rather than a structural alteration in the sensory pathway of the trigeminal complex that can be seasonal. The horse could serve as a natural animal model for humans with idiopathic trigeminal neuralgia.

Solubility of Haloether Anesthetics in Human and Animal Blood

Background: Anesthetic blood solubility predicts pharmacokinetics for inhaled agents and is essential for determination of blood anesthetic concentrations from end-tidal gas concentrations using Henrys Law. Though used to model anesthetic effects in humans, there are limited interspecies solubility comparisons that include modern haloethers. This study aimed to measure hematocrit-adjusted blood:gas anesthetic partition coefficients (&lgr;B:G) for desflurane, sevoflurane, isoflurane, and methoxyflurane in humans and animals. Methods: Whole blood was collected from 20 rats, 8 horses, and 4 each of cats, cattle, humans, dogs, goats, pigs, rabbits, and sheep. Plasma or cell volume was removed to adjust all samples to a packed cell volume of 40%. A single-agent calibration gas headspace was added to blood in a glass syringe and was mixed and equilibrated at 37°C for 2 h. Agent concentrations in the calibration gas and syringe headspace were measured using gas chromatography. Anesthetic solubility in saline, citrate-phosphate-dextrose-adenine, and olive oil were similarly measured. Results: Except for goats, all animal species had at least one &lgr;B:G measurement that differed significantly from humans. For each agent, &lgr;B:G positively correlated with serum triglyceride concentrations, but this only explained 25% of interspecies variability. Desflurane was significantly less soluble in blood than sevoflurane in some species (e.g., humans) but not in others (e.g., rabbits). Conclusions: Anesthetic partition coefficients differ significantly between humans and most animals for haloether anesthetics. Because of their similar &lgr;B:G values, goats may be a better animal model for inhaled anesthetic pharmacokinetics in people.

Chirality in anesthesia II: Stereoselective modulation of ion channel function by secondary alcohol enantiomers

Chirality has been proposed as a means for distinguishing relevant from irrelevant molecular targets of action, but the sensitivity and specificity of this test is unknown for volatile anesthetics. We applied enantiomers of two chiral anesthetic alcohols (2-butanol and 2-pentanol) that are enantioselective for the minimum alveolar concentration (MAC) preventing movement in 50% of animals and one (2-hexanol) that was not to frog oocytes. Each oocyte expressed one of three anesthetic-sensitive ion channels: a Twik-related-spinal cord K+ (TRESK) channel, a &ggr;-amino butyric acid type A (GABAA) receptor and an N-methyl-d-aspartate (NMDA) receptor. Using voltage-clamp techniques, we found that 2-butanol was not enantioselective for any channel (e.g., 16 mM 2-butanol R(−) and S(−) enantiomers decreased current through an NMDA receptors by 44% ± 3% [mean ± se] and 37% ± 4%, respectively); 2-pentanol was enantioselective for one channel (the GABAA receptor, the enantiomers increasing current by 277% ± 20% and 141% ± 30%); 2-hexanol was enantioselective for both GABAA and NMDA receptors (e.g., decreasing current through the NMDA receptor by 19% ± 3% and 43% ± 5%). We calculated the sensitivity and specificity of chirality as a test of anesthetic relevance under two scenarios: 1) all three channels were relevant mediators of MAC and 2) no channel was a mediator of MAC. These sensitivities and specificities were poor because there is no consistent correspondence between receptor and whole animal results. We recommend that enantioselectivity not be used as a test of relevance for inhaled anesthetic targets.