Microdialysis System Research Articles

Inflammation-Responsive Mesoporous Silica Nanoparticles with Synergistic Anti-inflammatory and Joint Protection Effects forRheumatoid Arthritis Treatment.

Joint destruction is a major burden and an unsolved problem in rheumatoid arthritis (RA) patients. We designed an intra-articular mesoporous silica nanosystem (MSN-TP@PDA-GlcN) with anti-inflammatory and joint protection effects. The nanosystem was synthesized by encapsulating triptolide (TP) in mesoporous silica nanoparticles and coating it with pH-sensitive polydopamine (PDA) and glucosamine (GlcN) grafting on the PDA. The nano-drug delivery system with anti-inflammatory and joint protection effects should have good potency against RA. A template method was used to synthesize mesoporous silica (MSN). MSN-TP@PDA-GlcN was synthesized via MSN loading with TP, coating with PDA and grafting of GlcN on PDA. The drug release behavior was tested. A cellular inflammatory model and a rat RA model were used to evaluate the effects on RA. In vivo imaging and microdialysis (MD) system were used to analyze the sustained release and pharmacokinetics in RA rats. TMSN-TP@PDA-GlcN was stable, had good biocompatibility, and exhibited sustained release of drugs in acidic environments. It had excellent anti-inflammatory effects in vitro and in vivo. It also effectively repaired joint destruction in vivo without causing any tissue toxicity. In vivo imaging and pharmacokinetics experiments showed that the nanosystem prolonged the residence time, lowered the Cmax value and enhanced the relative bioavailability of TP. These results demonstrated that MSN-TP@PDA-GlcN sustained the release of drugs in inflammatory joints and produced effective anti-inflammatory and joint protection effects on RA. This study provides a new strategy for the treatment of RA.

Multi-Region Microdialysis Imaging Platform Revealed Dorsal Raphe Nucleus Calcium Signaling and Serotonin Dynamics during Nociceptive Pain

In this research, we combined our ultralight micro-imaging device for calcium imaging with microdialysis to simultaneously visualize neural activity in the dorsal raphe nucleus (DRN) and measure serotonin release in the central nucleus of the amygdala (CeA) and the anterior cingulate cortex (ACC). Using this platform, we observed brain activity following nociception induced by formalin injection in the mouse's hind paw. Our device showed that DRN fluorescence intensity increased after formalin injection, and the increase was highly correlated with the elevation in serotonin release in both the CeA and ACC. The increase in calcium fluorescence intensity occurred during the acute and inflammatory phases, which suggests the biphasic response of nociceptive pain. Furthermore, we found that the increase in fluorescence intensity was positively correlated with mouse licking behavior. Lastly, we compared the laterality of pain stimulation and found that DRN fluorescence activity was higher for contralateral stimulation. Microdialysis showed that CeA serotonin concentration increased only after contralateral stimulation, while ACC serotonin release responded bilaterally. In conclusion, our study not only revealed the inter-regional serotonergic connection among the DRN, the CeA, and the ACC, but also demonstrated that our device is feasible for multi-site implantation in conjunction with a microdialysis system, allowing the simultaneous multi-modal observation of different regions in the brain.

Development of an integrated microperfusion-EEG electrode for unbiased multimodal sampling of brain interstitial fluid and concurrent neural activity

Objective. To modify off-the-shelf components to build a device for collecting electroencephalography (EEG) from macroelectrodes surrounded by large fluid access ports sampled by an integrated microperfusion system in order to establish a method for sampling brain interstitial fluid (ISF) at the site of stimulation or seizure activity with no bias for molecular size. Approach. Twenty-four 560 µm diameter holes were ablated through the sheath surrounding one platinum–iridium macroelectrode of a standard Spencer depth electrode using a femtosecond UV laser. A syringe pump was converted to push–pull configuration and connected to the fluidics catheter of a commercially available microdialysis system. The fluidics were inserted into the lumen of the modified Spencer electrode with the microdialysis membrane removed, converting the system to open flow microperfusion. Electrical performance and analyte recovery were measured and parameters were systematically altered to improve performance. An optimized device was tested in the pig brain and unbiased quantitative mass spectrometry was used to characterize the perfusate collected from the peri-electrode brain in response to stimulation. Main results. Optimized parameters resulted in >70% recovery of 70 kDa dextran from a tissue analog. The optimized device was implanted in the cortex of a pig and perfusate was collected during four 60 min epochs. Following a baseline epoch, the macroelectrode surrounded by microperfusion ports was stimulated at 2 Hz (0.7 mA, 200 µs pulse width). Following a post-stimulation epoch, the cortex near the electrode was stimulated with benzylpenicillin to induce epileptiform activity. Proteomic analysis of the perfusates revealed a unique inflammatory signature induced by electrical stimulation. This signature was not detected in bulk tissue ISF. Significance. A modified dual-sensing electrode that permits coincident detection of EEG and ISF at the site of epileptiform neural activity may reveal novel pathogenic mechanisms and therapeutic targets that are otherwise undetectable at the bulk tissue level.

Pharmacokinetics and transplacental transfer of codeine and codeine metabolites from Papaver somniferum L.

Ethnopharmacological relevancePapaveris Pericarpium, which is the dried husk of Papaver somniferum L., has been used as a phytomedicine to relieve cough, diarrhea and pain. The alkaloid codeine contained therein via biotransformation converts to morphine and potentially produces addictive and toxic effects. Due to the healthy concern for a pregnant woman, our hypothesis is that codeine and its metabolites can penetrate the placental barrier to reach the foetus and amniotic fluid, and these processes may be modulated by the transporter. Aim of the studyBecause codeine is also considered a prodrug of morphine, it has a good analgesic effect. It is often used by pregnant women but may expose the foetus to the risk of morphine harm. The aim of this study is to investigate the metabolic rate, distribution and transplacental transfer mechanism of codeine and its metabolites morphine and morphine-3-glucuronide (M3G) in pregnant rats and to assess the risk of medication for pregnant women. Materials and methodsUltrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) combined with a microdialysis system was developed to monitor codeine, morphine and M3G in multiple sites of maternal blood, placenta, foetus and amniotic fluid after codeine administration. A compartmental model was used to calculate the pharmacokinetic parameters of codeine in blood after codeine administration (10 mg/kg, i.v.). The area under the concentration (AUC) ratio of AUCmetabolite/AUCcodeine and AUCtissue/AUCblood was used to represent the metabolic biotransformation ratio and the drug from blood-to-tissue transfer ratio, respectively. ResultsThe pharmacokinetic results demonstrated that codeine fit well with a two-compartment model and went through rapid metabolism to morphine and M3G in pregnant rats after codeine administration (10 mg/kg, i.v.). The biotransformation ratios of AUCmorphine/AUCcodeine, AUCM3G/AUCmorphine and AUCM3G/AUCcodeine were 0.12 ± 0.03, 54.45 ± 20.61 and 6.53 ± 2.47, respectively, after codeine administration (10 mg/kg, i.v.), which suggested that codeine was easily metabolized into M3G through morphine. The tissue distribution results demonstrated that all of the analytes penetrated into the foetus through the placenta; however, the blood-to-tissue transfer ratio (AUCtissue/AUCblood) of morphine and M3G was relatively lower than that of codeine after codeine administration (10 mg/kg, i.v.), which suggested that the blood-placenta barrier blocks the penetration of morphine and M3G into the foetus. Thus, the tissue transfer of morphine in the placenta and foetus was significantly enhanced by treatment with corticosterone, an inhibitor of organic cation transporter (OCT). ConclusionBased on microdialysis coupled to a validated UHPLC–MS/MS system, the pharmacokinetics and metabolic biotransformation of codeine and its metabolites were analyzed and clarified. The potential mechanism of morphine placental transfer was modulated by OCT transporters.

Effect of the Size of Protein Therapeutics on Brain Pharmacokinetics Following Systematic Administration

Here, we have investigated the effect of size of protein therapeutics on brain pharmacokinetics (PK) following systemic administration in rats. All tested proteins were derived from trastuzumab that do not bind to any targets in rats. PK data generated with F(ab)2(100kDa), Fab (50kDa), and scFv (27kDa) fragments of trastuzumab, along with published PK data for FcRn non-binding and wild-type trastuzumab (150kDa), were used to establish a relationship between the protein size and brain exposure. A large-pore microdialysis system was used to measure the PK of proteins in the plasma, the interstitial fluid (ISF) at the striatum (ST), and the cerebrospinal fluid (CSF) at the lateral ventricle (LV) and cisterna magna (CM). Concentrations of all the proteins in plasma, brain homogenate, ISF, and CSF were measured using ELISA. When evaluating the effect of protein size in the absence of FcRn binding, we found a bell-shaped relationship between the size and ISF/plasma AUC ratio, where 100kDa F(ab)2 demonstrated the highest exposure. A similar bell-shaped relationship was observed for the brain homogenate/plasma AUC ratio, with a peak at 50kDa. The CSF/plasma AUC ratio at LV increased monotonously with a decrease in the size of proteins. We observed that the exposure of protein therapeutics in different regions of the brain could be significantly different and there could be optimal sizes of protein therapeutics to accomplish maximum/selective exposure in selected brain regions following systemic administration.

Trans-placental transfer of nicotine: Modulation by organic cation transporters.

Nicotine is a highly addictive substance and harmful to the developing foetus. However, few studies have investigated the transporter mechanism responsible for regulating the transfer of nicotine across the blood-placental interface. A multiple in-vivo microdialysis system coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to monitor simultaneously nicotine and cotinine in the blood, placenta, foetus, and amniotic fluid of pregnant rats. The pharmacological mechanism of nicotine transfer across the placenta was investigated by co-administering corticosterone, an inhibitor of organic cation transporters (OCTs) that partly mediate the exchange of nicotine across the placenta. The results revealed that intravenously administered nicotine (1mg/kg) was rapidly metabolised to cotinine with a transformation ratio (AUCcotinine/AUCnicotine) of 0.67±0.08, 0.21±0.05, 0.25±0.12, 0.31±0.05 in maternal blood, placenta, amniotic fluid, and foetus, respectively. The tissue transformation ratios (AUCtissue/AUCblood) were 0.83±0.16, 0.65±0.17, 0.57±0.13 for nicotine, and 0.25±0.06, 0.24±0.12, 0.26±0.04 for cotinine at placenta, amniotic fluid and foetus, respectively. Following the co-administration of corticosterone (2mg/kg), the tissue transformation ratio of nicotine was significantly reduced in the placenta but was significantly increased in the foetus. Levels of cotinine were not significantly altered by the administration of corticosterone. These findings implicate OCT in mediating the transfer of nicotine across the blood-placenta barrier. Understanding the mechanism of nicotine transfer through the placenta may inform therapeutic strategies to lessen the exposure of the developing foetus to nicotine in the maternal bloodstream.

Evaluation of Benzylpenicillin as an Internal Standard for Measurement of Piperacillin Bone Concentrations Via Microdialysis.

Microdialysis is a pharmacokinetic tool that can be advantageous when obtaining tissues' pharmacokinetic information. Since absolute extracellular tissue concentrations are needed in pharmacokinetic studies, calibrating the microdialysis system is necessary. The internal standard method is superior when compared to other calibration methods. However, thorough evaluation of the internal standard is required before it can be used. In vitro experiments and an in vivo study on pigs (n=8) were conducted to assess the relative recoveries by gain and by loss for piperacillin, both with and without a benzylpenicillin concentration of 5µg/mL. Furthermore, the in vivo setup allowed for an evaluation of piperacillin cancellous bone and subcutaneous tissue concentrations in a single 8h dosing interval. Ultra-high performance liquid chromatography (UHPLC) was used to determine piperacillin and benzylpenicillin concentrations. Relative recovery by loss for benzylpenicillin and relative recovery by gain for piperacillin were similar in in vitro and in vivo. Presence of benzylpenicillin did not affect the relative recovery for piperacillin. Relative recovery, pharmacokinetic parameters and fT>MIC were similar when comparing the retrodialysis by drug and the internal standard calibration methods (p > 0.31). Mean fT>MIC (16µg/mL) for plasma, cancellous bone and subcutaneous tissue were 232min, 255min and 295min, respectively. Our findings suggest that benzylpenicillin is suitable as an internal standard for piperacillin in microdialysis studies. Mean fT>MIC (16µg/mL) for plasma, cancellous bone, and subcutaneous tissue reached a target of 50% fT>MIC under the investigated conditions (mean range: 52%-66%); however, the target was not obtained in all pigs in all compartments. Moreover, 100% fT>MIC was not obtained in any case, suggesting that different strategies must be taken into consideration if higher targets are employed.

Organic nitrogen enhances nitrogen nutrition and early growth of Pinus sylvestris seedlings.

Boreal trees are capable of taking up organic nitrogen (N) as effectively as inorganic N. Depending on the abundance of soil N forms, plants may adjust physiological and morphological traits to optimize N uptake. However, the link between these traits and N uptake in response to soil N sources is poorly understood. We examined Pinus sylvestris L. seedlings’ biomass growth and allocation, transpiration and N uptake in response to additions of organic N (the amino acid arginine) or inorganic N (ammonium nitrate). We also monitored in situ soil N fluxes in the pots following an addition of N, using a microdialysis system. Supplying organic N resulted in a stable soil N flux, whereas the inorganic N resulted in a sharp increase of nitrate flux followed by a rapid decline, demonstrating a fluctuating N supply and a risk for loss of nitrate from the growth medium. Seedlings supplied with organic N achieved a greater biomass with a higher N content, thus reaching a higher N recovery compared with those supplied inorganic N. In spite of a higher N concentration in organic N seedlings, root-to-shoot ratio and transpiration per unit leaf area were similar to those of inorganic N seedlings. We conclude that enhanced seedlings’ nutrition and growth under the organic N source may be attributed to a stable supply of N, owing to a strong retention rate in the soil medium.

Blood-Placental Barrier Transfers and Pharmacokinetics of Unbound Morphine in Pregnant Rats with Multiple Microdialysis Systems.

Microdialysis coupled to an analytical system can be used to continuously monitor unbound protein analytes in any biological fluid, tissue, or organ of animals. To date, no application of microdialysis has been performed to simultaneously monitor unbound morphine and its metabolites in the placenta and fetus of pregnant rats. Our hypothesis is that morphine and its metabolite penetrate the blood-placental barrier to reach the fetus during pregnancy. To investigate this hypothesis, this study aimed to develop a microdialysis experimental animal model coupled with an analytical system to monitor morphine and morphine-3-glucuronide (M3G) in the maternal blood, placenta, fetus, and amniotic fluid of pregnant rats. To determine the analytes in dialysates, a validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed. The pharmacokinetic results indicated that morphine fit well to a two-compartment model and exhibited nonlinear pharmacokinetic behavior within the dosage regimen. The M3G-to-morphine metabolite ratio, determined by the area under the concentration curve (AUC) ratio (AUCM3G/AUCmorphine), was approximately 5.40 in the maternal blood. In terms of tissue distribution, the mother-to-fetus transfer ratio (AUCfetus/AUCblood) of morphine and M3G was about 0.34 and 0.18, respectively. In conclusion, the high metabolite ratio suggests that morphine has the characteristics of rapid biotransformation, and the mother-to-fetus transfer ratio indicates that morphine and M3G partially transfer the blood-placental barrier in pregnant rats. This newly developed multiple microdialysis coupled to UHPLC-MS/MS system can be applied to the studies of maternal pharmacokinetics and blood-placental transfer in pregnant rats.

Monitoring Neuronal Dynamics in the Ventral Tegmental Area Using an Implantable Microimaging Device With Microdialysis System

To monitor dopamine neural activities in the ventral tegmental area (VTA) in response to nicotine, we designed a dopamine platform based on a microimaging device and a microdialysis system. We measured dopamine (DA) release in the prefrontal cortex (PFC) and the nucleus accumbens (NAc) by a micro dialysis system and monitored DA neurons activities by fluorescence imaging in the VTA using a microimaging device simultaneously. GCaMP6 transgenic mice were used in this study, and the change in the fluorescence ratio intensity associated with nicotine administration was estimated. Our results suggested that nicotine administration increased the DA neurons activity in the VTA as well as the DA release in the PFC and the NAc. The proposed platform has the potential to monitor neural activities in the deep brain regions of rodents.

Transdermal delivery of triptolide–phospholipid complex to treat rheumatoid arthritis

The aim of this study was to develop and evaluate a triptolide phospholipid complex (TPCX) for the treatment of rheumatoid arthritis (RA) by transdermal delivery. TPCX was prepared and characterized by differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR) analysis, transmission electron microscope (TEM), and scanning electron microscope (SEM). The solubility of TPCX was determined. Then, a TPCX cream was prepared to evaluate its percutaneous permeability and the antiarthritis effect. The transdermal permeability was determined using the Franz method, and a microdialysis system was used for skin pharmacokinetic study. A rat model of RA was prepared to evaluate the pharmacological effects. TPCX increased the solubility of triptolide in water, and the percutaneous permeability of TPCX cream was greatly enhanced compared with triptolide cream. The skin pharmacokinetic study indicated that TPCX cream has a longer biological half-life (t 1/2) and mean residence time (MRT), but it has a shorter T max than that of triptolide cream in vivo. The area under the curve (AUC0– t )/AUC0–∞) and the peak concentration (C max) of TPCX cream were obviously higher than those of triptolide cream. The TPCX-loaded cream alleviated paw swelling and slowed down the progression of arthritis by inhibiting the inflammatory response by down regulating the TNF-α, IL-1β, and IL-6 levels, thus exhibiting excellent antiarthritic effects. In summary, the prepared TPCX effectively increases the hydrophilicity of triptolide, which is good for its percutaneous absorption and enhances its effect on RA rats. TPCX can be a good candidate for the transdermal delivery to treat RA.

Protein unbound pharmacokinetics of ambroxol in the blood and brains of rats and the interaction of ambroxol with Polygala tenuifolia by multiple microdialysis

Ethnopharmacological relevanceAmbroxol elevates glucocerebrosidase (GCase) activity and reduces nigrostriatal alpha-synuclein burden to better ameliorate motor function in Parkinson's disease (PD). Polygala tenuifolia is a potential alternative botanical medicine for the treatment of many nonmotor symptoms of PD commonly used in Taiwanese patients. Co-administration of these two medicines pose potential herb-drug interaction. Aim of the studyOur hypothesis is that ambroxol and P. tenuifolia may potentially possess herbal drug synergetic effects in the blood and brain. Materials and methodsTo investigate this hypothesis, a multiple microdialysis system coupled with validated ultra-high performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS) method was developed for rat blood and brain samples. Experimental rats were divided into three groups: low-dose and high-dose ambroxol alone (10 mg/kg, i.v. and 30 mg/kg, i.v., respectively) and ambroxol (10 mg/kg, i.v.) pretreated with P. tenuifolia extract (1 g/kg, p.o. for 5 consecutive days). ResultsAmbroxol easily penetrated into the brain and reached a maximum concentration in the striatum at approximately 60 min after low- and high-dose treatment. The area under the concentration curve (AUC) ratio increased proportionally at the doses of 10 and 30 mg/kg, which suggested a linear pharmacokinetic manner of ambroxol. The brain penetration of ambroxol was approximately 30–34%, which was defined as the ambroxol AUC blood-to-brain distribution ratio (AUCbrain/AUCblood). The P. tenuifolia extract did not significantly alter the pharmacokinetics of ambroxol in the blood and brain of rats. ConclusionThe present study suggests that it is safety without pharmacokinetic interactions for this dosing regimen to use P. tenuifolia extract and ambroxol together.

Sampling insulin in different tissue compartments using microdialysis: methodological aspects

Sampling the concentration of insulin in human skin using microdialysis is challenging because of low intracutaneous concentrations and low recovery, presumably due to adsorption of insulin to the microdialysis system. In this study, we aimed to (1) measure how the concentration of insulin varies in three different tissue compartments (intracutaneous, subcutaneous and intravenous) and (2) to study how much insulin is adsorbed to the microdialysis catheter membranes and tubing during a typical microdialysis experiment, both in vivo and in vitro. We hypothesized that (1) the concentration of insulin decreases from the intravenous compartment to the intracutaneous and subcutaneous tissue, and that (2) adsorption of insulin to the microdialysis membrane and tubing impairs the recovery of insulin from the tissue. In this experimental study, microdialysis catheters were inserted intracutaneously, subcutaneously and intravenously in 11 healthy subjects. Systemic endogenous hyperinsulinemia was induced by intake of an oral glucose load. Insulin concentration was measured in the dialysate and in the extracted samples from the catheter membrane and tubings. In vitro microdialysis was performed to investigate the temporal resolution of the adsorption. After an oral glucose load insulin concentration increased intravenously, but not in the intracutaneous or subcutaneous compartments, while glucose, lactate and pyruvate concentrations increased in all compartments. The adsorption of insulin to the microdialysis membrane in vivo was highest in the intravenous compartment (p = 0.01), compared to the intracutaneous and subcutaneous compartments. In vitro, the adsorption to the microdialysis membrane was highest one hour after sampling, then the concentration gradually decreased after three and five hours of sampling. The concentration of insulin in peripheral tissues is low, probably due to decreasing tissue vascularity. Adsorption of insulin to the microdialysis membrane is modest but time-dependent. This finding highlights the importance of a stabilization time for the microdialysis system before sampling tissue analytes.

Addressing the instability issue of dopamine during microdialysis: the determination of dopamine, serotonin, methamphetamine and its metabolites in rat brain.

Dopamine is a catecholamine neurotransmitter that degrades rapidly in aqueous solutions; hence, its analysis following brain microdialysis is challenging. The aim of the current study was to develop and validate a new microdialysis coupled LC-MS/MS system with improved accuracy, precision, simplicity and turnaround time for dopamine, serotonin, methamphetamine, amphetamine, 4-hydroxymethamphetamine and 4-hydroxyamphetamine analysis in the brain. Dopamine degradation was studied with different stabilizing agents under different storage conditions. The modified microdialysis system was tested in vitro, and was optimized for best probe recovery, assessed by %gain. LC-MS/MS assay was developed and validated for the targeted compounds. Stabilizing agents (ascorbic acid, EDTA and acetic acid) as well as internal and cold standards were added on-line to the dialysate flow. Assay linearity range was 0.01-100ng/mL, precision and accuracy passed criteria, and LOQ and LLOQ were 0.2 and 1.0 pg, respectively. The new microdialysis coupled LC-MS/MS system was used in Wistar rats striatum after 4mg/kg subcutaneous methamphetamine. Methamphetamine rapidly distributed to rat striatum reaching an average ~200ng/mL maximum, ~82.5min post-dose. Amphetamine, followed by 4-hydroxymethamphetamine, was the most abundant metabolite. Dopamine was released following methamphetamine injection, while serotonin was not altered. In conclusion, we proposed and tested an innovative and simplified solution to improve stability, accuracy and turnover time to monitor unstable molecules, such as dopamine, by microdialysis.

The Peacock study: feasibility of the dynamic characterisation of the paediatric hypothalamic-pituitary-adrenal function during and after cardiac surgery

BackgroundCortisol is the main stress hormone mobilised during surgery to establish homeostasis. Our current understanding of the hypothalamic-pituitary-adrenal axis physiology in children undergoing cardiopulmonary bypass is very limited due to: (1) very few cortisol time point measurements over long periods (2) difficulties of sampling in low weight babies and (3) the concomitant use of glucocorticoids at anaesthesia induction. This lack of understanding is reflected in a lack of consensus on the utility of glucocorticoids perioperatively in cardiac surgery with the use of cardiopulmonary bypass.MethodsThe Peacock Study is a prospective, two-centre, observational cohort study of 78 children (undergoing cardiopulmonary bypass procedures and non-surgical procedures - split by age/cyanosis) that aims to characterise in detail the hypothalamic-pituitary-adrenal axis physiology of children using the stress model of paediatric cardiac surgery. Also, we aim to correlate cortisol profiles with clinical outcome data. We herein describe the main study design and report the full cortisol profile of one child undergoing heart surgery, thus proving the feasibility of the method.ResultsWe used an automated, 24-h tissue microdialysis system to measure cortisol and cortisone, every 20 min. We herein report one cortisol profile of a child undergoing heart surgery. Besides, we measured serum cortisol and adrenocorticotrophic hormone at seven-time points for correlation. Tissue concentrations of cortisol increased markedly several hours after the end of surgery. We also noted an increase in the tissue cortisol/cortisone ratio during this response.ConclusionWe report for the first time, the use of an automated microdialysis sampling system to evaluate the paediatric adrenal response in children. Changes in cortisol and cortisone could be measured, and the concentration of cortisol in the tissues increased after the end of cardiac surgery. The method has wide application to measure other hormones dynamically and frequently without the limitation of the circulating blood volume. The data from the main study will clarify how these cortisol profiles vary with age, pathology, type of procedure and correlation to clinical outcomes.Trial registrationISCRTN registry, number: 982586.

SAT-639 Is the Freestyle Libre Flash Glucose Monitor Accurate in the Critically Ill?

The FreeStyle Libre flash glucose monitor (FGM) has made the use of continuous glucose monitors more accessible to the typical diabetes patient in an outpatient setting given the significantly lower cost and ease of use of FGM as compared to other systems. However, FGM is not labeled for use in a critically ill population. The critical care department at our institution queried the endocrine department about studying the use of FGM in critically ill patients. The interest of the critical care department was due to the potential of decrease in patient discomfort and decrease in time and effort of nursing and support staff related to the performance of fingerstick capillary glucoses if FGM was an adequate replacement measure.As of yet, there has been only minimal study of flash glucose monitoring in critically ill patients. One Australian study evaluated 8 patients in an ICU setting and determined that as compared with arterial blood glucose monitoring, flash glucose monitoring provided acceptable numerical and clinical accuracy.1 A Swedish study evaluated a total of 26 patients undergoing cardiac surgery and compared the use of FGM to use of a microdialysis intravascular system and concluded that the microdialysis system was more accurate, though in this study, only 25% of patients had diabetes. 2To further investigate use of FGM in a critically ill population, we plan to undertake a single center, prospective, single arm study enrolling at least 20 and up to 40 patients. Inclusion criteria include a known diagnosis of type 1 or type 2 diabetes, age of 18 or older, and admission to the medical intensive care unit (MICU) with expected MICU stay of at least 48 hours.Participating subjects will have a sensor applied by a study investigator. After confirmation that the sensor is operational, the investigator will place opaque tape over the monitor to blind the monitor data. Nurses or medical assistants will conduct the standard of care fingerstick glucose monitoring per hospital protocol but will also have been notified of request to also pass FGM reader over the sensor at time of fingerstick glucose data collection.The primary objectives are to determine numerical accuracy in a critical care setting using the mean absolute relative difference and to determine clinical accuracy in a critical care setting using the surveillance error grid and the clarke error grid analyses. Preliminary data should be available by March, 2020.1.Ancona P, Eastwood GM, Lucchetta L, Ekinci EI, Bellomo R, Martensson J. The performance of flash glucose monitoring in critically ill patients with diabetes. Crit Care Resusc 2017; 19: 167-174, June 2017.2.Schierenbeck F, Franco-Cereceda A, Liska J. Accuracy of 2 Different Continuous Glucose Monitoring Systems in Patients Undergoing Cardiac Surgery: Intravascular Microdialysis Versus Subcutaneous Tissue Monitoring. Journal of Diabetes Science and Technology 2017, Vol. 11(1) 108–116

The effect of continuous digital hypothermia on lamellar energy metabolism and perfusion during laminitis development in two experimental models.

Continuous digital hypothermia (CDH) prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp (EHC) and oligofructose (OF) laminitis models, but the mechanisms remain unclear. To evaluate the effects of CDH on lamellar energy metabolism and perfusion in healthy horses and during EHC and OF laminitis models. In vivo experiment. Archived samples were used from Standardbred geldings that received no treatment (CON) (n=8) or underwent EHC (n=8) or OF (n=6) laminitis models. Both forelimbs were instrumented with a lamellar microdialysis system, and one forelimb was cooled (CDH) with the other maintained at ambient temperature (AMB). Microdialysate was collected every 6hours and analysed for glucose, lactate and pyruvate concentrations and lactate to pyruvate ratio (L:P). Microdialysis urea clearance was used to estimate lamellar tissue perfusion. Data were analysed using a mixed-effects linear regression model. Glucose did not change in CDH limbs relative to AMB in CON (P=.3), EHC (P=.3) or OF (P=.6) groups. There was a decrease in lactate (P<.001) and pyruvate (P<.01) in CDH limbs relative to AMB in all groups. L:P decreased in CON CDH relative to CON AMB (P<.001) but was not different in EHC (P=.6) and OF (P=.07) groups. Urea clearance decreased in CDH limbs relative to AMB in CON (P=.002) and EHC (P<.001), but not in OF (P=.4). The EHC model may not mimic natural endocrinopathic laminitis. CDH caused a marked decrease in lamellar glucose metabolism (CON, EHC and OF) and perfusion (CON and EHC) without affecting lamellar glucose concentration. Although cellular energy failure is not a primary pathophysiological event in EHC and OF laminitis models, CDH may act by limiting energy supply to pathologic cellular processes whilst preserving those critical to lamellar homoeostasis.

Facile Ratiometric Electrochemical Sensor for In Vivo/Online Repetitive Measurements of Cerebral Ascorbic Acid in Brain Microdiaysate.

The in vivo monitoring of ascorbic acid (AA) following physiological and pathological events is of great importance because AA plays a critical role in brain functions. The conventional electrochemical sensors (ECSs) usually suffered from poor selectivity and sluggish electron transfer kinetics for cerebral AA oxidation. The exploitation of ECSs adapt to the electrochemical detection (ECD)-microdialysis system, here we reported a facile ratiometric electrochemical sensor (RECS) for in vivo/online repetitive measurements of cerebral AA in brain microdiaysate. The sensor were constructed by careful electrodeposition of graphene oxide (GO) onto glassy carbon (GC) electrodes. Methylene blue (MB) was electrostatically adsorbed onto the GO surface as a built-in reference to achieve ratiometric detection of AA. The subsequent proper electroreduction treatment was able to readily facilitate the oxidation of AA at a relatively negative potential (-100 mV) and the oxidation of MB at separated potential (-428 mV). The in vitro experiments demonstrated that the RECS exhibited high sensitivity (detection limit: 10 nM), selectivity, and stability toward AA determination, enabling the in vivo/online repetitive measurement of cerebral AA in brain microdiaysate with high reliability. As a result, the designed RECS was successfully applied in the ECD-microdialysis system to in vivo/online repetitive monitoring the dynamic change of cerebral AA in the progress of the global cerebral ischemia/reperfusion events. More, the microinjection of endogenous AA and AA oxidase (AAOx) verified the reliability of the proposed RECS for in vivo/online repetitive cerebral AA detection. This proposed sensor filled the gap that no rational electrochemical sensor has been developed for the ECD-microdialysis system since its creation by the Mao group in 2005, which provided a reliable and effective method for brain chemistry research.

Exclusion of unsuitable CNS drug candidates based on their physicochemical properties and unbound fractions in biomatrices for brain microdialysis investigations

Microdialysis has been the only direct method of continuously measuring the unbound drug concentrations in extracellular fluid at a specific brain region with respect to time in the same animal. However, not every compound is suitable for microdialysis system as demonstrated by their inconsistent “by gain” and “by loss” in-vitro microdialysis probe recoveries leading to over- or under- estimated in-vivo concentrations. Therefore, our current study was proposed aiming to develop simple exclusion criteria for drug candidates that are not suitable for microdialysis system investigation. Through literature research, the properties ((LogP, pKa, water solubility and unbound fraction in plasma and brain) of drugs that have been reported for microdialysis studies were summarized. The exclusion criteria were developed by evaluating the impact of such properties on the consistency of in-vitro “by gain” and “by loss” recoveries of microdialysis probe. As a result, forty-five compounds were identified from literatures, among which doxorubicin, docetaxel, omeprazole, donepezil and phenytoin were found to have inconsistent in-vitro “by gain” and “by loss” microdialysis probe recoveries and subsequently selected for the exclusion criteria analysis. It was found that compounds with limited water solubility (less than 1 g/L) and unbound fraction in plasma (fu,plasma less than 30%) and brain homogenate (fu,brain less than 10%) were more likely to have inconsistent “by gain” and “by loss” microdialysis probe recoveries. Our proposed exclusion criteria were further validated using carbamazepine (limited water solubility only), DB213 (limited fu,brain only) and piperine (both limited water solubility and limited fu,plasma, fu,brain). Our current proposed exclusion criteria will help excluding the CNS drug candidates that are highly unlikely suitable for brain microdialysis approach leading to a better success rate in brain microdialysis approach development.

In vitro evaluation of an intravenous microdialysis catheter for therapeutic drug monitoring of gentamicin and vancomycin.

A central venous catheter with a built‐in microdialysis membrane is available for continuous lactate and glucose monitoring in the intensive care unit (ICU). As this catheter might also be suitable for repeated measurements of unbound drug levels, we studied in vitro the feasibility of monitoring unbound antibiotic concentrations. The catheter was placed in various media at 37°C spiked with gentamicin or vancomycin. Dialysate fractions were repeatedly collected over 3 hours with a NaCl 0.9% perfusate flow of 5 μL/min. Total and unbound drug concentrations in medium and perfusate were measured by immunoassay. After 60 minutes stable recovery for both drugs was observed, with mean ±SD relative recoveries of vancomycin and gentamicin in human serum of 64% ±0.4% and 73% ±3%. The recoveries of the unbound concentrations were 91% ±3% and 91% ±4%. This intravenous microdialysis system may be a very useful platform for therapeutic drug monitoring in the ICU.