Mitochondrial membrane potential probes and the proton gradient: a practical usage guide

Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.     

Microdialysis for metabolic monitoring during septicemia]

Currently, metabolic changes in ICU patients in critical states are determined mainly by indirect laboratory parameters (e.g. blood lactate). Microdialysis is a new means of performing metabolic monitoring that permits organ-specific objectification on the basis of interstitial fluid samples. Continuous endotoxin infusion was administered to 10 female pigs and, in addition to hemodynamic monitoring, lactate and glycerol in the subcutaneous, intramuscular and hepatic tissue were measured by microdialysis. The interstitial concentrations of lactate and glycerol rose significantly under endotoxaemia and showed an earlier increase than blood lactate levels. Microdialysis is simple to apply, appears to be a suitable means of obtaining important information about cellular metabolic changes in different tissues of the critically ill patient, and can detect subtle changes that laboratory parameters can identify only later and incompletely.     

Slow non-specific accumulation of 2′-deoxy and 2′-O-methyl oligonucleotide probes at mitochondria in live cells

Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2′-deoxy and 2′-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.     

Microdialysis as a procedure for evaluating intestinal hypoxia--an animal experiment approach]

Usual ICU monitoring of patients with abdominal pathology provides no detailed information about hepatosplanchnic haemodynamics or intestinal metabolism. In our animal experiment, the effects of systemic hypoxia on microdialysis measurements of the peritoneum in comparison with the ischiocrural muscle as reference were investigated in 7 rats. The parameter of interest was the course of glucose metabolism reflecting sympathoadrenergic activity during the experiment. Measurements were obtained at timed intervals at baseline, under hypoxia, and during reoxygenation. After induction of systemic hypoxia, the peritoneal microdialysis showed significantly higher levels of glucose in comparison with the ischiocrural muscle. The results indicate hypermetabolic activity or a hypersympathetic response of the bowel in response to hypoxic stress. In the clinical setting, the bowel has an important role in the development of multiorgan failure. Microdialysis may therefore be an interesting tool for the early detection of hypoxic metabolism during and after abdominal procedures.     

Theory relating in vitro and in vivo microdialysis with one or two probes

In this paper, we further develop the general theory of microdialysis by extending the linear model of Bungay et al. to provide a theoretical basis for in vitro and in vivo microdialysis. Specifically, we considered the effect of active clearance processes on in vivo microdialysis, and thereby elaborated the theory of Benveniste et al. to endogenous compounds. We examined the use of steady state tissue diffusion resistance with negligible clearance processes to interpret microdialysis data. The influence of the tissue properties on the in vitro and in vivo recoveries in dual-probe microdialysis was analyzed and we simulated the effect of the operating parameters on dual probe microdialysis performance. We estimated that the minimum clearance rate constant detectable by microdialysis in a quasi-steady state is about 5.5 x 10(-5) s(-1). This minimum rate constant establishes a criterion, below which inhibition of the active clearance processes does not show detectable influences on the microdialysis extraction efficiency.     

微透析技术在脑靶向中应用

微透析技术作为一门新兴的技术,近年来多用于靶向分布和体内代谢等方面,尤其是在药物的脑部研究方面,该技术显得尤为重要。如今,随着新型探针的不断出现,以及微量、快速、灵敏的分析检测手段的发展,微透析技术已日益成为药物脑部研究的重要工具。现通过检索近十年来的相关文献,对脑微透析技术的概况、原理、脑微透析探针以及其应用作一综述,希望能为从事该方面研究的药学工作者提供相关参考。     

Utilization of in vivo ultrafiltration in biomedical research and clinical applications

Ultrafiltration (UF) is a filtrate selection method with a wide range of biomedical and clinical applications, including detoxification of blood in hemodialysis and peritoneal dialysis. New is, however, the use of UF as a convenient in vivo sampling method that, for example, has been used in diabetics. Ultrafiltration avoids complicated and time-consuming recovery calculations that are necessary when using in vivo microdialysis, as recoveries of low molecular weight molecules are near 100%. The subcutaneously or intravenously placed UF probes have been studied for off-line sample analysis and for continuous on-line monitoring, in a wide variety of species, including dogs, rats, pigs and humans. This review discusses the potential of in vivo UF as a continuous tissue sampling technique in clinical research areas, and in several major biomedical applications including glucose and lactate monitoring and drug kinetic studies.     

中国野生动物红外相机监测与研究: 现状及未来

智能传感器、人工智能、信息技术等现代科学技术的创新应用极大地提升了人类在全球生物多样性保护和恢复方面的潜力。结合国内外相关研究案例, 本文的主要内容包括: (1)对过去30年间(1991-2021年)中国野生动物红外相机监测研究相关文献资料进行总结分析; (2)结合国内2011年以来的典型案例, 对技术方法、物种发现与编目、形态与行为研究、生态学研究和保护管理等主题领域的进展进行总结分析; (3)结合国外近期的典型案例, 对红外相机监测与研究的重点领域进行评估分析; (4)对中国野生动物红外相机监测研究的未来发展提出相关建议。通过回顾, 本文旨在明晰国内外红外相机技术在野生动物监测研究中的创新应用和发展趋势, 为中国在该领域的未来发展提供参考依据, 以便更好地服务于中国生物多样性监测与研究网络建设和以国家公园为主体的自然保护地体系建设, 为推进国家生态文明建设、保障生态安全和生物安全提供决策支持和科学依据。     

Direct Comparison of the Response of Voltammetry and Microdialysis to Electrically Evoked Release of Striatal Dopamine

Abstract: Carbon fiber microelectrodes either were implanted directly into striatal tissue or were mounted into the outlet of microdialysis probes that were implanted into striatal tissue. This allowed voltammetry and microdialysis to be used under identical in vivo experimental conditions to monitor extracellular dopamine levels during electrical stimulation of the medial forebrain bundle both before and after uptake inhibition with nomifensine. The marked differences between the results obtained with each technique cannot be explained on the basis of their inherent analytical attributes (sensitivity, temporal response, etc.). The results demonstrate that the microdialysis recovery factor for endogenous dopamine increases after uptake inhibition, an observation that stands in contradiction to the existing theory and practice of the microdialysis technique. The observations led to the development of a numerical model that rationalizes the observations reported herein and that allows in vivo voltammetry and in vivo microdialysis results to be interpreted within a single theoretical framework.     

Brain microdialysis and its applications in experimental neurochemistry

Microdialysis is one of the most powerful neurochemistry techniques, which allows the monitoring of changes in the extracellular content of endogenous and exogenous substances in the brain of living animals. The strength as well as wide applicability of this experimental approach are based on the bulk theory of brain neurotransmission. This methodological review introduces basic principles of chemical neurotransmission and emphasizes the difference in neurotransmission types. Clear understanding of their significance and degree of engagement in regulation of physiological processes is an ultimate prerequisite not only for choosing an appropriate method of monitoring for interneuronal communication via chemical messengers but also for accurate data interpretation. The focus on the processes of synthesis/metabolism, receptor interaction/ neuronal signaling or the behavioral relevance of neurochemical events sculpts the experiment design. Brain microdialysis is an important method for examining changes in the content of any substances, irrespective of their origin, in living animals. This article compares contemporary approaches and techniques that are used for monitoring neurotransmission (including in vivo brain microdialysis, voltammetric methods, etc). We highlight practical aspects of microdialysis experiments in particular to those researchers who are seeking to increase the repertoire of their experimental techniques with brain microdialysis.     

Experimental and theoretical microdialysis studies of in situ metabolism

Microdialysis sampling was performed to monitor localized metabolism in vivo and in vitro. A mathematical model that accounts for analyte mass transport during microdialysis sampling was used to predict metabolite concentrations in the microdialysis probe during localized metabolism experiments. The model predicts that metabolite concentrations obtained in the microdialysis probe are a function of different experimental parameters including membrane length, perfusion fluid flow rate, and sample diffusive and kinetic properties. Different microdialysis experimental parameters including membrane length and perfusion fluid flow rate were varied to affect substrate extraction efficiency (E(d)), or loss to the sample matrix, in vivo and in vitro. Local hepatic metabolism was studied in vivo in male Sprague-Dawley rats by infusing acetaminophen through the microdialysis probe. Acetaminophen sulfate concentrations increased linearly with respect to acetaminophen E(d) in contrast to modeling predictions. Xanthine oxidase was used as an in vitro model of localized metabolism. In vitro experimental results partially matched modeling predictions for 10-mm probes. These results suggest that monitoring local metabolism using microdialysis sampling is feasible. It is important to consider system parameters such as dialysis flow rate, membrane length, and sample properties because these factors will affect analyte concentrations obtained during local metabolism experiments.     

Coupled Effects of Mass Transfer and Uptake Kinetics on In Vivo Microdialysis of Dopamine

Abstract: Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug-naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high-affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.     

Validation of real-time RT-PCR assays for mRNA quantification in baboons

Real-time RT-PCR has been used widely, both in fundamental research and in clinical diagnostics, for instance for quantification of RNA levels in human tissues and tissue biopsies. In the present study we provide a strategy to validate primers/probes for real-time RT-PCR quantification of baboon samples. The method is based on the TaqMan system and uses primers/probes that have been designed and validated for human real-time RT-PCR. A prerequisite for the accuracy of this strategy is a similar amplification efficiency between human and baboon PCR reactions. We propose two different methods, i.e. by calculating PCR efficiencies from the slope of a dilution curve or by using the linear regression method, to compare the amplification efficiency between human and baboon samples. In conclusion, by performing a simple validation experiment, real-time PCR assays based on human sequences, which are easily available, can be applied for analysis of baboon samples.     

Microdialysis: intraoperative and posttraumatic applications in neurosurgery

Microdialysis offers a unique opportunity to study the extracellular human brain environment. Our aim was to improve the ability to detect neuronal injury in patients undergoing complicated neurovascular procedures and to detect secondary brain lesions after severe head injury with the use of in vivo microdialytic monitoring. We employed intraoperative microdialysis monitoring in patients with a variety of neurovascular procedures including high-flow and low-flow bypass surgery and aneurysm clipping. In the second group there were patients after severe brain injury where in vivo microdialysis was performed as a bedside monitoring in the intensive care unit. In this review we present our results for these various groups of patients, in which a variety of neurochemical parameters which are known to change in ischemia were studied. The advantages and disadvantages of the technique are discussed. Our results show that microdialysis is a useful tool for detecting ischemic changes intraoperatively and in severe head trauma. However, further studies are needed to get more data and to better refine the online microdialysis technique before we can recommend it for routine clinical use.     

Roots‐eye view: Using microdialysis and microCT to non‐destructively map root nutrient depletion and accumulation zones

Improvement in fertilizer use efficiency is a key aspect for achieving sustainable agriculture in order to minimize costs, greenhouse gas emissions, and pollution from nutrient run‐off. To optimize root architecture for nutrient uptake and efficiency, we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling, such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values that can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non‐invasive, continuous method for sampling available nutrients in the soil. Here, for the first time, we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots whereas ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root‐available nutrients non‐destructively and in almost real time.     

环境DNA在两栖动物监测中的应用研究进展

两栖动物是我国受威胁程度最高的动物类群,加强两栖动物资源调查和多样性监测,是开展两栖动物保护和濒危物种拯救行动的关键性基础工作。传统的两栖动物监测主要以形态学和声学为基础,耗时费力,且难以发现一些隐蔽性较强的稀有物种。基于环境DNA(environmental DNA, eDNA)的调查方法以其快速、灵敏、高效、无创等独特优势,为两栖动物多样性监测及保护提供了新的工具。综述了eDNA在两栖动物多样性监测、外来入侵和珍稀濒危物种调查、物种丰度或生物量估测等研究领域的应用进展,分析了两栖动物eDNA产生、扩散、迁移和降解的动态变化特征及其关键影响因子,探讨了eDNA应用于两栖动物监测研究的局限性并提出了优化建议,同时对未来的研究方向进行了展望,以充分挖掘eDNA在两栖动物监测中的应用潜力,为两栖动物多样性保护和管理提供新的思路。     

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter''s experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.     

Striatal Tissue Preparation Facilitates Early Sampling in Microdialysis and Reveals an Index of Neuronal Damage

Abstract: Injury-induced efflux of dopamine was compared between two microdialysis preparations. Rats were implanted with guide cannulae 5–10 days prior to microdialysis experiments. In one group, ventral striatal tissue was punctured with stainless steel obturators that remained in place until the day of the experiment. In the other group, the tissue was not punctured until the microdialysis probes were inserted. Rats from each group were dialyzed with calcium-free artificial extracellular fluid or tetrodotoxin 4 h after probe insertion. In the rats with previously punctured tissue, calcium depletion reduced dialysate dopamine concentrations to 8% of baseline. Dialysis with tetrodotoxin reduced dopamine concentrations to less than 1% of baseline. In the rats with freshly punctured tissue, dopamine concentrations were reduced only to 50% of baseline levels by calcium depletion and to 30% during dialysis with tetrodotoxin. Thus, penetration of the tissue prior to testing can significantly reduce the acute injury-induced efflux of dopamine. Further, a significant correlation was found between baseline 3,4-dihydroxy-phenylacetic acid/dopamine ratios and the efficacy of tetrodotoxin in reducing dialysate dopamine concentrations. Thus, basal 3,4-dihydroxyphenylacetic acid/dopamine ratios appear to provide an index of the amount of injury-induced dopamine efflux following probe insertion.