Free interstitial levels of metformin in the liver of healthy and diabetic Wistar rats

Abstract In the present study, free interstitial levels reached by metformin in the liver were investigated in control and diabetic rats by microdialysis. Firstly, a bioanalytical method using an HPLC-UV system to determine the drug concentration in microdialysis samples was validated. The blood glucose levels and biochemical parameters were investigated in control and diabetic animals. Following that, both groups received a dose of 50 mg/kg of metformin iv bolus and the free interstitial levels reached in the liver were assessed by microdialysis. The method was validated according to FDA guidelines being suitable to quantify free concentrations of metformin in the liver of control and diabetics rats. Free exposure to metformin was similar in control and diabetic animals: AUC0-∞ 118.50 ± 40.18 vs 112.93 ± 50.25 µg.h/mL, respectively. The half-life in tissue was similar to that described in the literature for plasma. Hence diabetes induced by streptozotocin after administration of nicotinamide in our study did not damage the renal and hepatic function of the animals. The levels reached in the liver were 1.6 times higher than the free plasma concentrations, demonstrating higher liver penetration of metformin. This is the first investigation in liver interstitial concentration of metformin in control and diabetic rats

Microdialysis and microperfusion electrodes in neurologic disease monitoring.

Contemporary biomarker collection techniques in blood and cerebrospinal fluid have to date offered only modest clinical insights into neurologic diseases such as epilepsy and glioma. Conversely, the collection of human electroencephalography (EEG) data has long been the standard of care in these patients, enabling individualized insights for therapy and revealing fundamental principles of human neurophysiology. Increasing interest exists in simultaneously measuring neurochemical biomarkers and electrophysiological data to enhance our understanding of human disease mechanisms. This review compares microdialysis, microperfusion, and implanted EEG probe architectures and performance parameters. Invasive consequences of probe implantation are also investigated along with the functional impact of biofouling. Finally, previously developed microdialysis electrodes and microperfusion electrodes are reviewed in preclinical and clinical settings. Critically, current and precedent microdialysis and microperfusion probes lack the ability to collect neurochemical data that is spatially and temporally coincident with EEG data derived from depth electrodes. This ultimately limits diagnostic and therapeutic progress in epilepsy and glioma research. However, this gap also provides a unique opportunity to create a dual-sensing technology that will provide unprecedented insights into the pathogenic mechanisms of human neurologic disease.

Cerebral Microdialysis as a Tool for Assessing the Delivery of Chemotherapy in Brain Tumor Patients.

The development of curative treatment for glioblastoma has been extremely challenging. Chemotherapeutic agents that have seemed promising have failed in clinical trials. Drugs that can successfully target cancer cells within the brain must first traverse the brain interstitial fluid. Cerebral microdialysis (CMD) is an invasive technique in which interstitial fluid can be directly sampled. CMD has primarily been used clinically in the setting of head trauma and subarachnoid hemorrhage. Our goal was to review the techniques, principles, and new data pertaining to CMD to highlight its use in neuro-oncology. We conducted a literature search using the PubMed database and selected studies in which the investigators had used CMD in either animal brain tumor models or clinical trials. The references were reviewed for additional information. Studies of CMD have shown its importance as a neurosurgical technique. CMD allows for the collection of pharmacokinetic data on drug penetrance across the blood-brain barrier and metabolic data to characterize the response to chemotherapy. Although no complications have been reported, the current CMD technique (as with any procedure) has risks and limitations, which we have described in the present report. Animal CMD experiments have been used to exclude central nervous system drug candidates from progressing to clinical trials. At present, patients undergoing CMD have been monitored in the intensive care unit, owing to the requisite tethering to the apparatus. This can be expected to change soon because of advances in microminiaturization. CMD is an extremely valuable, yet underused, technique. Future CMD applications will have central importance in assessing drug delivery to tumor cells in vivo, allowing a pathway to successful therapy for malignant brain tumors.

Development of a highly sensitive gas chromatography-mass spectrometry method preceded by solid-phase microextraction for the analysis of propofol in low-volume cerebral microdialysate samples.

To date, the commonly used intravenous anesthetic propofol has been widely studied, and fundamental pharmacodynamic and pharmacokinetic characteristics of the drug are known. However, propofol has not yet been quantified in vivo in the target organ, the human brain. Here, cerebral microdialysis offers the unique opportunity to sample propofol in the living human organism. Therefore, a highly sensitive analytical method for propofol quantitation in small sample volumes of 30 µL, based on direct immersion solid-phase microextraction was developed. Preconcentration was followed by gas chromatographic separation and mass spectrometric detection of the compound. This optimized method provided a linear range between the lower limit of detection (50 ng/L) and 200 µg/L. Matrix-matched calibration was used to compensate recovery issues. A precision of 2.7% relative standard deviation between five consecutive measurements and an interday precision of 6.4% relative standard deviation could be achieved. Furthermore, the permeability of propofol through a cerebral microdialysate system was tested. In summary, the developed method to analyze cerebral microdialysate samples, allows the in vivo quantitation of propofol in the living human brain. Additionally the calculation of extracellular fluid levels is enabled since the recovery of the cerebral microdialysis regarding propofol was determined.

Proteomic analysis and ATP assay reveal a positive effect of artificial cerebral spinal fluid perfusion following microdialysis sampling on repair of probe-induced brain damage.

BACKGROUND: Microdialysis (MD) is conventionally used to measure the in vivo levels of various substances and metabolites in extracellular and cerebrospinal fluid of brain. However, insertion of the MD probe and subsequent perfusion to obtain samples cause damage in the vicinity of the insertion site, raising questions regarding the validity of the measurements. NEW METHOD: We used fluorogenic derivatization liquid chromatography-tandem mass spectrometry, that quantifies both high and low abundance proteins, to differentiate the effects of perfusion from the effects of probe insertion on the proteomic profiles of expressed proteins in rat brain. RESULTS: We found that the expression levels of five proteins were significantly lower in the perfusion group than in the non-perfusion group. Three of these proteins are directly involved in ATP synthesis. In contrast to decreased levels of the three proteins involved in ATP synthesis, ATP assays show that perfusion, following probe insertion, even for a short time (3 h) increased ATP level up to 148% that prior to perfusion, and returned it to normal state (before probe insertion). COMPARISON WITH EXISTING METHOD: There is essentially no information regarding which observed changes are due to probe insertion and which to perfusion. CONCLUSIONS: Our findings partially demonstrate that the influence of whole MD sampling process may not significantly compromise brain function and subsequent analytical results may have physiological equivalence to normal, although energy production is transiently damaged by probe insertion.

Assessment of changes in blood glucose concentration with intravascular microdialysis.

Blood glucose and its variability of is a major prognostic factor associated with morbidity. We hypothesized that intravenous microdialysis incorporated in a central venous catheter (CVC) would be interchangeable with changes in blood glucose measured by the reference method using a blood gas analyzer. Microdialysis and central venous blood glucose measurements were simultaneously recorded in high-risk cardiac surgical patients. The correlation between absolute values was determined by linear regression and the Bland-Altman test for repeated measurements was used to compare bias, precision, and limits of agreement. Changes in blood glucose measurement were evaluated by four-quadrant plot and trend interchangeability methods (TIM). In the 23 patients analyzed, the CVC was used as part of standard care with no complications. The correlation coefficient for absolute values (N = 99) was R = 0.91 (P < 0.001). The bias, precision and limits of agreement were - 9.1, 17.4 and - 43.2 to 24.9 mg/dL, respectively. The concordance rate for changes in blood glucose measurements (N = 77) was 85% with the four-quadrant plot. The TIM showed that 14 (18%) changes of blood glucose measurements were uninterpretable. Among the remaining 63 (82%) interpretable changes, 23 (37%) were interchangeable, 13 (20%) were in the gray zone, and 27 (43%) were not interchangeable. Microdialysis using a CVC appears to provide imprecise absolute blood glucose values with risk of insulin misuse. Moreover, only one third of changes in blood glucose measurements were interchangeable with the reference method using the TIM.

Pharmacokinetics of doxorubicin in glioblastoma multiforme following ultrasound-Induced blood-brain barrier disruption as determined by microdialysis.

The goal of this study was to investigate the in vivo extracellular kinetics of doxorubicin (Dox) in glioblastoma multiforme (GBM)-bearing mice following focused ultrasound (FUS)-induced blood-brain barrier (BBB) disruption using microdialysis. An intracranial brain tumor model in NOD-scid mice using human brain GBM 8401 cells was used in this study. Prior to each sonication, simultaneous intravenous administration of Dox and microbubbles, and the Dox concentration in the brains was quantified by high performance liquid chromatography (HPLC). Drug administration with sonication elevated the tumor-to-normal brain Dox ratio of the target tumors by about 2.35-fold compared with the control tumors. The mean peak concentration of Dox in the sonicated GBM dialysate was 10 times greater than without sonication, and the area under the concentration-time curve was 3.3 times greater. This study demonstrates that intracerebral microdialysis is an effective means of evaluating real-time target BBB transport profiles and offers the possibility of investigating the pharmacokinetics of drug delivery in the sonicated brain.

Assessment of changes in lactate concentration with intravascular microdialysis during high-risk cardiac surgery using the trend interchangeability method.

Background: Blood lactate is a strong predictor of mortality, and repeated blood lactate assays are recommended during surgery in high-risk patients. We hypothesized that the use of intravascular microdialysis incorporated in a central venous catheter would be interchangeable with the reference blood gas technique to monitor changes in blood lactate. Methods: Microdialysis and central venous blood lactate measurements were recorded simultaneously in high-risk cardiac surgical patients. The correlation between absolute values was determined by linear regression, and the Bland-Altman test for repeated measurements was used to compare bias, precision, and limits of agreement. Changes in lactate measurements were evaluated with a four-quadrant plot and trend interchangeability method (TIM). Results: In the 23 patients analysed, the central venous catheter was used as part of standard care, with no complications. The correlation coefficient for absolute values ( n =104) was 0.96 ( P <0.0001). The bias, precision, and limits of agreement were -0.19, 0.51, and -1.20 to 0.82 mmol litre -1 , respectively. The concordance rate for changes in blood lactate measurements ( n =80) was 94% with the four-quadrant plot. In contrast, the TIM showed that 23 (29) changes in lactate measurements were not interpretable, and among the remaining 57 (71) interpretable changes, 18 (32) were interchangeable, 8 (14) were in the grey zone, and 31 (54) were not interchangeable. Conclusions: Microdialysis with a central venous catheter appears to provide reliable absolute blood lactate values. Although changes in blood lactate measurements showed an excellent concordance rate, changes between the two methods were poorly interchangeable with the TIM. Clinical trial registration: NCT02296593.

Rapid Staining of Circulating Tumor Cells in Three-Dimensional Microwell Dialysis (3D-µDialysis) Chip.

The conventional techniques to detect circulating tumour cells (CTCs) are lengthy and the use of centrifugal forces in this technique may cause cell mortality. As the number of CTCs in patients is quite low, the present study aims towards a gentler diagnostic procedure so as not to lose too many CTCs during the sample preparation process. Hence, a Three-Dimensional Microwell dialysis (3D-µDialysis) chip was designed in this study to perform gentle fluorescence-removal process by using dialysis-type flow processes without centrifuging. This leads to a minimum manual handling of CTCs obtained in our study without any contamination. In addition, a rapid staining process which necessitates only about half the time of conventional techniques (35 minutes instead of 90 minutes) is being illustrated by the employment of dialysis process (by dynamically removing water and waste at once) instead of only static diffusion (by statically removing only waste by diffusion). Staining efficiency of our technique is improved over conventional staining because of the flow rate in 3D-µDialysis staining. Moreover, the staining process has been validated with clinical whole blood samples from three TNM stage IV colon cancer patients. The current technique may be termed as "miniature rapid staining and dialysing system".

Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes.

Development of new principles and methods for cerebral ATP assay is highly imperative not only for determining ATP dynamics in brain but also for understanding physiological and pathological processes related to ATP. Herein, we for the first time demonstrate that micrometer scale ion current rectification (MICR) at a polyimidazolium brush-modified micropipette can be used as the signal transduction output for the cerebral ATP assay with a high selectivity. The rationale for ATP assay is essentially based on the competitive binding ability between positively charged polyimidazolium and ATP toward negatively charged ATP aptamer. The method is well responsive to ATP with a good linearity within a concentration range from 5 nM to 100 nM, and high selectivity toward ATP. These properties essentially enable the method to determine the cerebral ATP by combining in vivo microdialysis. The basal dialysate level of ATP in rat brain cortex is determined to be 11.32 ± 2.36 nM (n = 3). This study demonstrates that the MICR-based sensors could be potentially used for monitoring neurochemicals in cerebral systems.

Microdialysis as a New Technique for Extracting Phenolic Compounds from Extra Virgin Olive Oil.

The amount and composition of the phenolic components play a major role in determining the quality of olive oil. The traditional liquid-liquid extraction (LLE) method requires a time-consuming sample preparation to obtain the "phenolic profile" of extra virgin olive oil (EVOO). This study aimed to develop a microdialysis extraction (MDE) as an alternative to the LLE method to evaluate the phenolic components of EVOO. To this purpose, a microdialysis device and dialysis procedure were developed. "Dynamic-oil" microdialysis was performed using an extracting solution (80:20 methanol/water) flow rate of 2 µL min-1 and a constant EVOO stream of 4 µL min-1. The results indicated a strong positive correlation between MDE and the LLE method, providing a very similar phenolic profile obtained with traditional LLE. In conclusion, the MDE approach, easier and quicker in comparison to LLE, provided a reliable procedure to determine the phenolic components used as a marker of the quality and traceability of EVOO.

Accuracy of 2 Different Continuous Glucose Monitoring Systems in Patients Undergoing Cardiac Surgery.

BACKGROUND: Continuous glucose monitoring (CGM) is today provided by various techniques. This study aims to compare two different CGM-systems: the FreeStyle Libre subcutaneous continuous glucose monitoring system (SC-CGM) and the Eirus intravascular microdialysis continuous glucose monitoring system (MD-CGM) in patients undergoing cardiac surgery. METHODS: A total of 26 patients were equipped with both the SC-CGM and the MD-CGM systems. The SC-CGM system was placed subcutaneously in the left upper-arm and the MD-CGM system was placed in the superior vena cava. Reference blood glucose values were obtained by analyzing arterial blood in a blood gas analyzer. Reference glucose values were then paired with glucose values from both CGM-systems and analyzed for accuracy. RESULTS: In all, 514 paired MD-CGM/arterial blood gas glucose values and 578 paired SC-CGM/arterial blood gas glucose values were obtained. Mean difference (SD) for the MD-CGM system was 0.9 (15.1) mg/dl and for the SC-CGM system -43.4 (20) mg/dl. ISO criteria (ISO15197:2013) were not met by either CGM system. In the Clarke error grid, all paired samples were within the zones AB for the MD-CGM system, and 94% in zone A. For the SC-CGM system, 99.1% of the paired samples were within zones AB, and 18.9% in zone A. Both the MD-CGM and the SC-CGM systems were reliable and used without complications. CONCLUSIONS: These results indicate that the Eirus intravascular microdialysis system monitors glucose continuously with superior accuracy compared to the FreeStyle Libre subcutaneous glucose monitoring system, which repeatedly measured a glucose value that was lower than the reference method.

In vivo online magnetic resonance quantification of absolute metabolite concentrations in microdialysate.

In order to study metabolic processes in animal models of diseases and in patients, microdialysis probes have evolved as powerful tools that are minimally invasive. However, analyses of microdialysate, performed remotely, do not provide real-time monitoring of microdialysate composition. Microdialysate solutions can theoretically be analyzed online inside a preclicinal or clinical MRI scanner using MRS techniques. Due to low NMR sensitivity, acquisitions of real-time NMR spectra on very small solution volumes (µL) with low metabolite concentrations (mM range) represent a major issue. To address this challenge we introduce the approach of combining a microdialysis probe with a custom-built magnetic resonance microprobe that allows for online metabolic analysis (1H and 13C) with high sensitivity under continuous flow conditions. This system is mounted inside an MRI scanner and allows performing simultaneously MRI experiments and rapid MRS metabolic analysis of the microdialysate. The feasibility of this approach is demonstrated by analyzing extracellular brain cancer cells (glioma) in vitro and brain metabolites in an animal model in vivo. We expect that our approach is readily translatable into clinical settings and can be used for a better and precise understanding of diseases linked to metabolic dysfunction.

Intraabdominal microdialysis - methodological challenges.

Microdialysis is used for in vivo sampling of extracellular molecules. The technique provides a continuous and dynamic view of concentrations of both endogenous released and exogenous administered substances. Microdialysis carries a low risk of complications and has proven to be a safe procedure in humans. The technique has been applied in several clinical areas, including gastrointestinal surgery. Microdialysis may be used for studies of tissue metabolism, and the technique is also a promising tool for pharmacological studies of drug penetration into abdominal organ tissue and the peritoneal cavity. The clinical significance of intraabdominal microdialysis in postoperative monitoring of surgical patients has yet to be proven. In this review, we introduce the microdialysis technique, and we present an overview of theoretical and practical considerations that should be taken into account when using microdialysis in intraabdominal clinical research.

Surface microdialysis on small bowel serosa in monitoring of ischemia.

BACKGROUND: Ischemic injury of an organ causes metabolic change from aerobic to anaerobic metabolism. It has been shown in experimental studies on the heart and liver that such conversion may be detected by conventional microdialysis probes placed intra-parenchymatously, as well as on organ surfaces, by assaying lactate, pyruvate, glucose, and glycerol in dialysate. We developed a microdialysis probe (S-µD) intended for use solely on organ surfaces. The aim of this study was to assess whether the newly developed S-µD probe could be used for detection and monitoring of small bowel ischemia. METHODS: In anesthetized normoventilated pigs, a control S-µD probe was applied on the jejunal serosa 50 cm downstream from the duodenojejunal junction (DJJ). Starting 100 cm from DJJ, a 100-cm long ischemic segment was created by division of all mesenteric vessels. S-µDs were applied at 2.5, 5, 20, and 50 cm from the starting point of ischemia by serosal sutures. A standard µD probe was placed in the abdominal cavity as a further control. Dialysate was harvested before inducing ischemia and subsequently every 20 min for 4 h. Central venous blood was drawn every hour to monitor systemic lactate, C-reactive protein, and white blood cell count. RESULTS: Microdialysis lactate levels were significantly higher than baseline from 20 min on into protocol time in the ischemic segment and in the control S-µD probe. The peritoneal cavity probe showed no significant elevation. Lactate levels from the ischemic segment reached a plateau at 60 min. Courses of pyruvate, glucose, and glycerol levels were in accordance with transition from an aerobic to anaerobic metabolism in the bowel wall. No statistically significant changes in hemoglobin, white blood cell count, or lactate values in central venous blood were recorded. CONCLUSIONS: Assaying the aforementioned compounds in dialysate, harvested by the newly developed S-µD probe, allowed detection and monitoring of small bowel ischemia from 20 min on following its onset.

Observational Evaluations of Mice during Cerebral Microdialysis for Pediatric Brain Tumor Research.

In vivo animal experiments are critical in the process of finding and developing new treatments for children with CNS tumors. Cerebral microdialysis, which enables researchers to measure drug concentrations in the brain or tumor tissue of unanesthetized mice, is a highly specialized procedure that provides valuable information that cannot be gained by using an in vitro system. When designing any in vivo animal study, 3 Rs principles (replacement, reduction, and refinement) must be considered to ensure that the highest standards of care are followed. As part of the refinement process, the objectives of this study were to collect behavioral monitoring data from mice undergoing cerebral microdialysis, to identify any behaviors predictive of significant pain or distress that could affect the animal's welfare, and to use these data to refine the existing monitoring checklist and schedule for its use by others performing this procedure. We developed a monitoring checklist for assessing wellbeing and distress of mice during cerebral microdialysis experiments. Comparison of 79 mice that underwent cerebral microdialysis experiments with a control group of 20 mice revealed that cerebral microdialysis and tethering of mice are well tolerated for as long as 24 h with only minor evidence of stress.

Intrabronchial Microdialysis: Effects of Probe Localization on Tissue Trauma and Drug Penetration into the Pulmonary Epithelial Lining Fluid.

Recent intrabronchial microdialysis data indicate that the respiratory epithelium is highly permeable to drugs. Of concern is whether intrabronchial microdialysis disrupts the integrity of the respiratory epithelium and thereby alters drug penetration into the pulmonary epithelial lining fluid (PELF). The objective of this study was to investigate the effect of intrabronchial microdialysis on the integrity of the bronchial epithelium. Microdialysis sampling in PELF in proximal (n = 4) and distal bronchi (n = 4) was performed after intravenous inulin and florfenicol administration in anaesthetized pigs. Inulin was used as a marker molecule of permeability of the epithelium, and florfenicol was used as test drug. Bronchial tissue was examined by histopathology (distal and proximal bronchi) and gene expression analysis (RT-qPCR, proximal bronchi) at the termination of the experiment (6.5 hr). The microdialysis probe caused overt tissue trauma in distal bronchi, whereas no histopathological lesions were observed in proximal bronchi. A moderate up-regulation of the pro-inflammatory cytokines IL1B, IL6 and acute-phase reactant serum amyloid A was seen in proximal bronchi surrounding the microdialysis probes suggesting initiation of an inflammatory response. The observed up-regulation is considered to have limited impact on drug penetration during short-term studies. Inulin penetrated the respiratory epithelium in both proximal and distal bronchi without any correlation to histopathological lesions. Likewise, florfenicol penetration into PELF was unaffected by bronchial histopathology. However, this independency of pathology on drug penetration may not be valid for other antibiotics. We conclude that short-term microdialysis drug quantification can be performed in proximal bronchi without disruption of tissue integrity.

Simultaneous determination of the repertoire of classical neurotransmitters released from embryonal carcinoma stem cells using online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry.

Dynamic, continuous, and simultaneous multi-analysis of transmitters is important for the delineation of the complex interactions between the neuronal and intercellular communications. But the analysis of the whole repertoire of classical transmitters of diverse structure is challenging due to their different physico-chemical properties and to their high polarity feature which leads to poor retention in traditional reversed-phase columns during LC-MS analysis. Here, an online microdialysis coupled with hydrophilic interaction chromatography-tandem mass spectrometry (online MD-HILIC-MS/MS) detection method was developed for the simultaneous measurement of the repertoire of classical transmitters (acetylcholine, serotonin, dopamine, norepinephrine, glutamate, GABA, and glycine). Stable isotope labeled internal standards and authentic matrix have been applied to guarantee reliable results. The method was successfully employed to reveal the characteristics of transmitter release from embryonal carcinoma stem cells. The method features simple procedure (no sample preparation), high recovery (≥ 73%), high accuracy (89.36%≤RE≤116.89%), good reproducibility (2.18%≤ RSD ≤14.56%), and sensitive limits of detection (2 pg for acetylcholine, serotonin, and glutamate, 10 pg for dopamine, norepinephrine, GABA, and glycine). It can be flexibly applied to determine the contents of the classical transmitters in other biological matrix samples with minor changes.

Self-built microdialysis probes with improved recoveries of ATP and neuropeptides.

BACKGROUND: Microdialysis is an established technique for collecting small molecular weight substances (e.g. neurotransmitter and energy metabolites) from the extracellular space. The major element of microdialysis is the probe which contains a semi-permeable membrane and is exposed to the interstitial space. As the microdialysis technique has major advantages, e.g. versatility and use in awake animals, commercially produced probes are in great demand. NEW METHOD: We here present the design of a probe assembly step by step which will enable researchers to build custom-made probes. Probe recoveries of substances with different molecular weight (ranging from 100 to 1600 Da) were compared for three different probes (CMA 12 Elite probe, custom-made 10 kDa and 30 kDa probes). Recoveries of glucose, lactate, acetylcholine, choline, ATP and the neuropeptides angiotensin II, substance P and somatostatin are presented. RESULTS: We found that the 10 kDa probe is only useful for compounds up to 1000 Da while recoveries of the CMA-12 Elite Probe are variable and apparently dependent on ionic charges of analytes. The recovery of the custom-made 30 kDa probe is highest and evidently not influenced by physicochemical parameters of analytes. In a further optimization step, we describe the use of ZipTip(®) µC-18 collection tips to replace the outlet tubing when purifying the dialysate for MALDI-MS measurements of neuropeptides. COMPARISON WITH EXISTING METHODS: The results show that self-built microdialysis probes can be equally or more effective than commercially available probes. CONCLUSIONS: Self-built microdialysis probes with large pore-membranes are capable of dialyzing ATP and neuropeptides.