High performance liquid chromatography-tandem mass spectrometric assay of dexmedetomidine in plasma, urine and amniotic fluid samples for pregnant ewe model.

Dexmedetomidine (DEX; Precedex(®)), approved by the Food and Drug Administration (FDA) in 1999 as a sedative for use in the intensive care unit, is a potent and highly selective α2-adrenoceptor agonist with significant sedative, analgesic and anxiolytic effects. However, the research of DEX use during pregnancy is limited and the impact of DEX on the fetal development is unclear. This article describes a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay suitable for various biomatrices of plasma, urine and amniotic fluid, as a prerequisite for pharmacokinetic characterization of DEX in the pregnant ewe model. DEX and testosterone (internal standard; IS) were extracted from 200µL of plasma, urine or amniotic fluid with ethyl acetate. The HPLC resolution was achieved on an Agilent ZORBAX SB-CN column with a gradient elution at a flow rate of 0.5mL/min using a mobile phase of 5-100% of acetonitrile with 0.5% formic acid (mobile phase B) in water (mobile phase A). The detection was performed by a triple quadrupole tandem mass spectrometer with positive electrospray ionization. The precursor/product transitions (m/z) in the positive ion mode [M+H](+) were m/z 201.5→95.4 for DEX and m/z 289.2→109.1 for IS. The method was validated in the concentration range of 25 (lower limit of quantification; LLOQ)-5000pg/mL for both maternal and fetal plasma, and of 50 (LLOQ)-5000pg/mL for urine and amniotic fluid, respectively. The intra- and inter-day precision and accuracy were within ±9%. The overall recoveries of DEX were 82.9-87.2%, 85.7-88.4%, 86.2-89.7% and 83.7-88.1% for maternal plasma, urine, fetal plasma and amniotic fluid, respectively. The percentage matrix factors in different biomatrices were less than 120%. Stability studies demonstrated that DEX was stable after three freeze/thaw cycles, in the autosampler tray at 20°C for 24h and during the 3h sample preparation at room temperature. The validated HPLC-MS/MS method has been successfully employed for pharmacokinetic evaluation of DEX in pregnant ewes and fetuses.

Fetal lung volume and quantification of liver herniation by magnetic resonance imaging in isolated congenital diaphragmatic hernia.

OBJECTIVE: To determine associations between fetal lung and liver herniation volumes measured by magnetic resonance imaging (MRI) and mortality/need for extracorporeal membrane oxygenation (ECMO) in cases of isolated congenital diaphragmatic hernia (CDH). A secondary objective was to compare prenatal MRI parameters with two-dimensional ultrasound lung measurements. METHODS: A retrospective review of medical records of all fetuses with isolated CDH evaluated between January 2004 and July 2012 was performed. The following MRI parameters were measured at 20-32 weeks: observed/expected total fetal lung volume (o/e-TLV), predicted pulmonary volume (PPV), percentage of liver herniated into the fetal thorax (%LH) and the liver/thoracic volume ratio (LiTR). These were compared with the ultrasound-determined lung-to-head ratio (LHR) and the observed/expected LHR (o/e-LHR) in the same cohort. The predictive value of MRI and ultrasound parameters for mortality and the need for ECMO was evaluated by univariate, multivariate and factor analysis and by receiver-operating characteristics curves. RESULTS: Eighty fetuses with isolated CDH were evaluated. Overall mortality was 18/80 (22.5%). Two newborns died a few hours after birth. ECMO was performed in 29/78 (37.2%) newborns, with a survival rate of 48.3% (14/29). The side of the diaphragmatic defect was not associated with mortality (P = 0.99) or the need for ECMO (P = 0.48). Good correlation was observed among o/e-TLV, PPV, LHR and o/e-LHR as well as between %LH and LiTR (r = 0.89; P < 0.01); however, fetal lung measurements and measures of liver herniation were not correlated (all P > 0.05). All parameters were statistically associated with mortality or the need for ECMO. The best combination of measurements to predict mortality was o/e-TLV and %LH, with 83% accuracy. CONCLUSION: Mortality and the need for ECMO in neonates with isolated CDH can be best predicted using a combination of MRI o/e-TLV and %LH.

Acceleration of recovery after injury to the peripheral nervous system using ultrasound and other therapeutic modalities.

Taken together, these studies show the promise of various therapeutic modalities for the noninvasive treatment of peripheral nerve injury. Further progress on these promising methods requires determining the biologic mechanisms responsible for the ability of these modalities to enhance peripheral nerve recovery. Necessary investigations include validation or refutation of the hypothesis that these therapies act on various aspects of the natural healing process. Examples include cellular and molecular processes involved in promoting Wallerian degeneration and the rate and specificity of axonal regeneration and remyelination and muscle reinnervation, processes that are distributed between the regenerating nerve itself, the pathway of the regenerating axon, and the target of the regenerating nerve. An increased understanding of the biologic mechanisms underlying the enhancement of peripheral nerve recovery after injury would lend greater insight into the cellular and molecular mechanisms involved in successful nerve regeneration and muscle reinnervation. This increased understanding may also result in clinically beneficial treatments for peripheral nerve disorders.

Analgesia elicited by OFQ/nociceptin and its fragments from the amygdala in rats.

The heptadecapeptide, orphanin FQ/nociceptin (OFQ/N), binds with high affinity to the ORL-1/KOR-3 opioid receptor clone, yet binds poorly with traditional opioid receptors. OFQ/N has a complex functional profile with relation to nociceptive processing, displaying pro-nociceptive properties in some studies, acting as an inhibitor of stress-induced analgesia in others, yet producing both spinal and supraspinal antinociceptive actions in other studies. Among the intracerebral sites at which OFQ/N might produce one or more of these actions is the amygdala which has been intimately implicated in both antinociceptive and stress-related responses. Therefore, the present study assessed whether microinjections into the amygdala of equimolar doses of OFQ/N(1-17) or its shorter-chained active fragments, OFQ/N(1-11) or OFQ/N(1-7), would produce analgesia as measured by either reactivity to high-intensity radiant heat or reactivity to electric shock, and produce hyperalgesia as measured by reactivity to lower-intensity radiant heat. OFQ/N(1-17) in the amygdala produced a dose-dependent and time-dependent increase in high-intensity tail-flick latencies with maximal effects observed at a dose range of 0.75-3 nmol, and lesser effects at lower (0.015-0.15 nmol) and higher (5.5-30 nmol) doses. Both OFQ/N(1-11) and OFQ/N(1-7) in the amygdala displayed lower magnitudes of analgesia than OFQ/N(1-17) on this measure, with OFQ/N(1-11) displaying maximal effects at higher (15-30 nmol) doses and OFQ/N(1-7) displaying maximal effects at lower (0.15-1.5 nmol) doses. In contrast to traditional mu and kappa opioids and beta-endorphin, none of the OFQ/N fragments in the amygdala exhibited any analgesic responses on the jump test. Finally, using a low-intensity radiant heat assay capable of detecting hyperalgesic responses, each of the OFQ/N fragments in the amygdala increased tail-flick latencies on this measure. Therefore, OFQ/N fragments appear to exert only analgesic responses in the amygdala with quantitative and qualitative differences relative to traditional opioid agonists.

Ultrasound accelerates functional recovery after peripheral nerve damage.

OBJECTIVE: Axonal injury in the peripheral nervous system is common, and often it is associated with severe long-term personal and societal costs. The objective of this study is to use an animal model to demonstrate that transcutaneous ultrasound can accelerate recovery from an axonotmetic injury. METHODS: The sciatic nerve of adult male Lewis rats was crushed in the right midthigh to cause complete distal degeneration of axons yet maintain continuity of the nerve. Beginning 3 days after surgery, various transcutaneous ultrasound treatments or sham treatments were applied 3 days per week for 30 days to the crush site of rats that were randomly assigned to two groups. In the preliminary experiments, there were three animals in each ultrasound group and two control animals. In the final experiment, there were 22 animals in the ultrasound group and 20 animals in the control group. Recovery was assessed by use of a toe spread assay to quantify a return to normal foot function in the injured leg. Equipment included a hand-held transducer that emitted continuous-wave ultrasound. The most successful ultrasound protocol had a spatial peak, time-averaged intensity of 0.25 W/cm2 operated at 2.25 MHz for 1 minute per application. RESULTS: Rats subjected to the most successful ultrasound protocol showed a statistically significant acceleration of foot function recovery starting 14 days after injury versus 18 days for the control group. Full recovery by the ultrasound group occurred before full recovery by the control group. CONCLUSION: Transcutaneous ultrasound applied to an animal model of axonotmetic injury accelerated recovery. Future studies should focus on identification of the mechanism(s) by which ultrasound creates this effect, as a prelude to optimization of the protocol, demonstration of its safety, and its eventual application to humans.

Modulation of macrophage and microglial responses to axonal injury in the peripheral and central nervous systems.

OBJECTIVE: After axonal injury, macrophages rapidly infiltrate and become activated in the mammalian peripheral nervous system (PNS) but not the central nervous system (CNS). We used the dorsal root pathway to study factors that modulate the response of macrophages to degenerating axons in both the PNS and the CNS. METHODS: Lewis rats underwent transection of dorsal roots (Group 1), stab within the spinal cord (Group II), crush at the dorsal root entry zone (Group III), transection of dorsal roots combined with a CNS lesion (Group IV), or systemic administration of a known activator of macrophages, lipopolysaccharide, alone (Group V) or combined with transection of dorsal roots (Group VI). ED-1 antibody stained for macrophages and activated microglia at 7, 14, and 42 days postinjury. RESULTS: At early time points, Group I demonstrated ED-1 cells in the PNS but not the CNS portion of the degenerating dorsal roots. Group II revealed ED-1 cells near the stab lesion. Group III demonstrated ED-1 cells adjacent to the dorsal root entry zone crush site. Group IV revealed ED-1 cells along both the PNS and the CNS portions of the degenerating dorsal roots when the CNS lesion was placed near the transected roots. Group V demonstrated few ED-1 cells in the PNS and the CNS, whereas Group VI revealed a marked ED-1 cellular response along both the PNS and the CNS portions of the transected dorsal roots. CONCLUSION: Local CNS trauma and systemic administration of lipopolysaccharide can "prime" macrophages/microglia, resulting in an enhanced response to degenerating axons in the CNS. Such priming might prove useful in promoting axonal regeneration.

Enhancement of adenosine A1 receptor functions by benzoylthiophenes in guinea pig tissues in vitro.

Previous reports on a series of benzoylthiophenes, including PD 81,723 [2-amino-4,5-dimethyl-3-(3-trifluoromethyl-benzoyl) thiophene], have shown specific enhancement of agonist binding at the adenosine A1 receptor. We have studied the effects of two substituted benzoylthiophenes, PD 78,416 (thieno[2,3-c]pyridine-6(5H)-carboxylic acid, 2-amino-3-benzoyl-4,7-dihydro-ethyl ester) and RS-74513-000 [2-amino-4-ethyl-5-methyl-3-(3-trifluoro-methyl-benzoyl) thiophene] on response elicited by adenosine A1 receptors in isolated guinea pig left atrium and ileum. In the electrically paced left atrium, PD 78,416 antagonized negative inotropic effect elicited by the agonist CPA [N6-cyclopentyladenosine] with a pKB value of 6.2 +/- 0.2 (n = 4). At a low concentration which had no antagonistic effect (0.1 microM), PD 78,416 enhanced the effect of CPA. The concentration-response curve to CPA was shifted leftward by 5.1 fold (95% confidence limits 2.4-11.2). In field stimulated isolated ileum, PD 78,416 (0.1, 0.3, 1 microM) did not enhance or antagonize effects of CPA. At concentrations above 1 microM, PD 78,416 decreased electrically induced contraction. This effect was not sensitive to adenosine deaminase and was not antagonized by the A1 antagonist CPX [8-cyclopentyl-1,3-dipropyl-xanthine] (1 microM). Unlike PD 78,416, RS-74513-000 (0.01, 0.1, 1, 3, 10 microM) did not antagonize or enhance effects of CPA in the left atrium. However, effects of CPA in ileum were enhanced by RS-74513-000 (1 and 3 microM).(ABSTRACT TRUNCATED AT 250 WORDS)