Early Detection of Metabolic Changes Using Microdialysis During and After Experimental Kidney Transplantation in a Porcine Model
Hamidreza FonouniMajid EsmaeilzadehParvin JarahianMorva Tahmasbi RadMohammad GolrizAlireza FaridarMohammadreza HafeziShadi JafariehArash KashfiSeid Hashem Fani YazdiMehrdad SoleimaniThomas LongerichM. Yu. ShevchenkoOliver SakowitzJan SchmidtArianeb Mehrabi
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Background: Microdialysis (MD) can detect organ-related metabolic changes before they become measurable in plasma through the biochemical parameters. This study aims to evaluate the early detection of metabolic changes during experimental kidney transplantation (KTx). Material and methods: During preparation of 8 donor kidneys, one MD catheter was inserted in the renal cortex and samples were collected. After a 6-hour cold ischemia time (CIT), kidneys were implanted in the 8 recipient pigs. Throughout the warm ischemia time (WIT) and after reperfusion, kidneys were monitored. The interstitial glucose, lactate, pyruvate, glutamate, and glycerol concentrations were evaluated. Results: A significant decline in glucose level was observed at the end of CIT. The lactate level was reduced to the minimum point of 0.35 ± 0.08 mmol/L in CIT. After reperfusion, lactate values raised significantly. During the WIT, the pyruvate level increased, continued until the end of the WIT. For glutamate, a steady increase was noted during explantation, CIT, WIT, and early reperfusion phases. The increase of glycerol value continued in the early postreperfusion, which was then followed by a sharp decline. Conclusion: MD is a fast and simple minimally invasive method for measurement of metabolic substrates in renal parenchyma during KTx. MD offers the option of detecting minor changes of interstitial glucose, lactate, pyruvate, glutamate, and glycerol in every stage of KTx. Through the use of MD, metabolic changes can be continuously monitored during the entire procedure of KTx.Keywords:
Microdialysis
Parenchyma
Machine Perfusion
Interstitial fluid
Microdialysis
Interstitial fluid
Jugular vein
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In this study we aimed to validate the microdialysis technique for metabolic measurements in the dermal interstitial fluid. The abdominal and forearm skin was used for microdialysis in 15 healthy normal weight volunteers. The depth of the microdialysis catheter was assessed by ultrasound measurement. Structural impairment and blood flow were judged from biopsies and from laser Doppler measurements taken adjacent to the catheters. Dermal interstitial lactate and pyruvate concentrations were measured, under steady state fasting conditions, after equilibrium calibration of each catheter in situ. The dermal interstitial glucose concentration was estimated by means of the retrodialysis calibration method, which has previously not been evaluated for skin microdialysis. The mean catheter depth (+/- standard deviation) was 0.8 +/- 0.3 mm. Small areas of localized bleeding, but no inflammatory reaction, was found surrounding the catheters. The perfusion in the microdialysis region was slightly increased (15-25%). The lactate/pyruvate ratio (12 +/- 0.7) showed non-ischaemic values. The dermal interstitial lactate concentration was significantly higher (1171 +/- 228 mumol/l) than the plasma lactate (781 +/- 180 mumol/l), indicating an ongoing nonoxidative glucose metabolism. Retrodialysis calibration correctly estimated the dermal glucose level to be similar to that in plasma, which may indicate the usefulness of this calibration method for microdialysis studies of endogenous substrates in the dermal interstitial fluid.
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Interstitial fluid
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Aims To investigate uptake of fluconazole into the interstitial fluid of human subcutaneous tissue using the microdialysis and suction blister techniques. Methods A sterile microdialysis probe (CMA/60) was inserted subcutaneously into the upper arm of five healthy volunteers following an overnight fast. Blisters were induced on the lower arm using gentle suction prior to ingestion of a single oral dose of fluconazole (200 mg). Microdialysate, blister fluid and blood were sampled over 8 h. Fluconazole concentrations were determined in each sample using a validated HPLC assay. In vivo recovery of fluconazole from the microdialysis probe was determined in each subject by perfusing the probe with fluconazole solution at the end of the 8 h sampling period. Individual in vivo recovery was used to calculate fluconazole concentrations in subcutaneous interstitial fluid. A physiologically based pharmacokinetic (PBPK) model was used to predict fluconazole concentrations in human subcutaneous interstitial fluid. Results There was a lag‐time (approximately 0.5 h) between detection of fluconazole in microdialysate compared with plasma in each subject. The in vivo recovery of fluconazole from the microdialysis probe ranged from 57.0 to 67.2%. The subcutaneous interstitial fluid concentrations obtained by microdialysis were very similar to the unbound concentrations of fluconazole in plasma with maximum concentration of 4.29 ± 1.19 µg ml −1 in subcutaneous interstitial fluid and 3.58 ± 0.14 µg ml −1 in plasma. Subcutaneous interstitial fluid‐to‐plasma partition coefficient (K p ) of fluconazole was 1.16 ± 0.22 (95% CI 0.96, 1.35). By contrast, fluconazole concentrations in blister fluid were significantly lower ( P < 0.05, paired t ‐test) than unbound plasma concentrations over the first 3 h and maximum concentrations in blister fluid had not been achieved at the end of the sampling period. There was good agreement between fluconazole concentrations derived from microdialysis sampling and those estimated using a blood flow‐limited PBPK model. Conclusions Microdialysis and suction blister techniques did not yield comparable results. It appears that microdialysis is a more appropriate technique for studying the rate of uptake of fluconazole into subcutaneous tissue. PBPK model simulation suggested that the distribution of fluconazole into subcutaneous interstitial fluid is dependent on tissue blood flow.
Microdialysis
Interstitial fluid
Suction blister
Subcutaneous tissue
Subcutaneous injection
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Interstitial microdialysis is a minimally invasive method that allows biochemical monitoring of endogenous and exogenous substances in extracellular tissue space. The method is based on sampling of soluble molecules from the interstitial space fluid by means of a semipermeable membrane. Microdialysis has been applied in primary and clinical research of metabolic changes and the blood flow in tissue interstitium and pharmacokinetic drug studies. Results obtained by microdialysis are gradually finding their place in clinical practice as well. This paper is aimed at presenting to the reader new technique of monitoring interstitial metabolism, its advantages, drawbacks and relevance for medicine.
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Interstitial fluid
Interstitial space
Semipermeable membrane
Clinical Practice
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NeuroPhage is developing NPT001 (filamentous bacteriophage M13) for the treatment of AD. NPT001 binds Aß with high affinity and disaggregates Aß plaque in a concentration-dependent manner in vitro and in vivo. In aged Tg2576 mice, amyloid plaque is significantly reduced after intra hippocampal injection of NPT001. The current study was conducted to determine if NPT001-induced clearance of Aß alters brain interstitial fluid (ISF) Aß levels. Microdialysis was used to assess ISF Aß in the brains of awake Tg2576 mice following intra hippocampal injection of NPT001. This technique measures a specific pool of unbound Aß that diffuses across a 38kDa MWCO membrane on the microdialysis probe. Aged female B6; SJL-Tg(APPSWE)2576Kha (Tg2576) mice were implanted with a unilateral 38-kDa MWCO microdialysis/injection probe into the hippocampus. Following recovery, six 90-min samples of microdialysis perfusion buffer were obtained to establish basal ISF Aß levels in each mouse. After basal sampling, 2ul of NPT001 or vehicle was injected into the hippocampus via the injection port at the tip of the microdialysis probe. ISF Aß samples were obtained across 5 days at 90-min increments following injection. Microdialysis samples were analyzed for Aßx-40 and Aßx-42 by sandwich ELISA. Following sampling, hippocampal Aß plaque load was quantified. ISF Aßx-40 levels did not increase significantly in NPT001-injected mice (n = 6) compared to vehicle-injected controls (n = 5). Interestingly, a slight lowering of ISF Aßx-40 fluctuating levels in NPT001-injected mice was observed across days. In a subset of mice tested (n = 2), Aßx-42 levels also did not increase following NPT001 injection. Importantly, NPT001 significantly reduced Aß plaque load by 52.5 ± 7.5% (mean ± SEM) in the injected hemisphere compared to the contralateral hemisphere in the same mice. In contrast, vehicle-injected mice had similar plaque load in the ipsilateral and contralateral hemispheres. All six mice in the NPT001-injected group had a decline in plaque load. Results indicate that ISF Aß levels, as assessed by in vivo microdialysis, did not change as a result of NPT001 injection despite a 50% reduction in Aß plaque load. NPT001 reduces Aß plaque load by a mechanism or pathway that does not involve solubilization and/or release of Aß monomers into brain ISF.
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Interstitial space
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<i>Background:</i> Uremic toxins are typically measured in plasma and little is known of their interstitial concentrations. We undertook experiments to validate a microdialysis technique for simultaneous recovery of small and large uremic toxins in the subcutaneous interstitial fluid (ISF). <i>Methods:</i> Microdialysis catheters were inserted into the subcutaneous interstitium of 8 subjects (controls and uremic patients) and perfused using two different solutions at incremental flow rates to determine analyte recovery and ISF concentrations of urea and protein. <i>Results:</i> 10% dextran-40 perfusate allowed the determination of interstitial concentrations of urea and protein reliably, by virtue of the exponential decay of their concentrations in the microdialysate with incremental flow rates (R<sup>2</sup> = 0.63–0.99). Interstitial and plasma urea correlated well (r = 0.95), as did interstitial urea from distant anatomical sites (r = 0.96). <i>Conclusion:</i> Cutaneous microdialysis with dextran-40 allows measurement of small and large molecule concentrations in ISF, creating an opportunity to characterize ISF in uremia.
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Interstitial fluid
Uremia
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According to the requirement of the calibration in minimally invasive blood glucose monitoring, a method based on microdialysis was presented to monitor glucose level in interstitial fluid continuously. An experimental system simulating the continuous change of glucose concentration in vivo was built. The influences on recovery of microdialysis caused by flow rate, glucose concentration, and temperature etc. were studied. The results led to the conclusion that the recovery fell by 71.7% when perfusion rate increased from 0.3 μL/min to 3.0 μL/min, while the different concentrations of glucose solutions scarcely contribute to the recovery instead, and the temperatures from 25 to 58 °C caused the recovery to increase by 34.6%.
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Interstitial fluid
Blood glucose monitoring
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Soluble amyloid-β (Aβ) peptide converts to structures with high β-sheet content in Alzheimer's disease (AD). Soluble Aβ is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Aβ levels may provide unique insights into Aβ metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Aβ microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Aβ in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Aβ deposition, significant changes in the ISF Aβ 40 /Aβ 42 ratio developed without changes in Aβ 1-x . These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Aβ production, we found that ISF Aβ half-life was short (∼2 hr) in young mice but was twofold longer in mice with Aβ deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Aβ synthesis reveals a portion of the insoluble Aβ pool that is in dynamic equilibrium with ISF Aβ. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.
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Interstitial fluid
Ex vivo
Amyloid (mycology)
Human brain
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To identify a perfusion flow at which the interstitial fluid completely equilibrates with the microdialysis perfusion fluid, a protocol with successively lower perfusion flows was used. A colloid was included in the perfusion fluid to make sampling possible at the lowest perfusion flows. At 0.16 μl/min, the measured metabolites had reached a complete equilibration in both tissues, and the measured concentrations of glucose, glycerol, and urea were in good agreement with expected tissue-specific levels. The glucose concentration in adipose tissue (4.98 ± 0.14 mM) was equal to that of plasma (5.07 ± 0.07 mM), whereas the concentration in muscle (4.41 ± 0.11 mM) was lower than in plasma and adipose tissue ( P < 0.001). The concentration of lactate was higher ( P< 0.001) in muscle (2.39 ± 0.22 mM) than in adipose tissue (1.30 ± 0.12 mM), whereas the glycerol concentration in adipose tissue (233 ± 19.7 μM) was higher ( P< 0.001) than in muscle (40.8 ± 3.0 μM) and in plasma (68.7 ± 3.97 μM). The concentration of urea was equal in the two tissues. Overall, the study indicates that microdialysis at a low perfusion flow may be a tool to continuously monitor tissue interstitial concentrations.
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Interstitial fluid
Muscle tissue
Interstitial space
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In vivo calibration of microdialysis catheters with [ 3 H]glucose as internal reference was done in rat ( n = 17) and human ( n = 12) subcutaneous tissue. The estimated interstititial glucose level was compared owth the glucose concentration in venous plasma which, in turn, has been shown to be identical to the interstitial glucose concentration. In subcutaneous tissue of anaesthetized male Sprageue‐Dawley rats, interstitial glucose was significantly overestimated (43%, P < 0.005, n = 8, and 19%, P < 0.005, n =9, in nornoglycaemic and hyperglycaemic animals, respectively). Furthermore, fractional outflux of [ 3 H]glucose decreased continuously during prolonged perfusion of the microdialysis catheter. Incontrast, in human subcutaneous tissue microdialysed with two catheters, correct measurements of interstitial glucose could be achieved and the precision was comparableto that obtained with equilibration calibraton in vivo . The average relative error of the mean result of two catheters was 8.9% at a perfusate flow rate of 1 μL min ‐1 . It may be suggested that calibration in in vivo of microdialysis catheters with internal references may be used in human subcutaneous tissue. However, it is necessary to validate the calibration technique in each different tissue under reproducible experimental conditions since accumulation of the reference substance in the tissue may create artefactual results.
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Subcutaneous tissue
Interstitial fluid
Interstitial space
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