Vivo Microdialysis Research Articles

In-Depth Chemical Analysis of the Brain Extracellular Space Using In Vivo Microdialysis with Liquid Chromatography-Tandem Mass Spectrometry.

Metabolomic analysis of samples acquired in vivo from the brain extracellular space by microdialysis sampling can provide insights into chemical underpinnings of a given brain state and how it changes over time. Small sample volumes and low physiological concentrations have limited the identification of compounds from this compartment, so at present, we have scant knowledge of its composition. As a result, most in vivo measurements have limited depth of analysis. Here, we describe an approach to (1) identify hundreds of compounds in brain dialysate and (2) routinely detect many of these compounds in 5 μL microdialysis samples to enable deep monitoring of brain chemistry in time-resolved studies. Dialysate samples collected over 12 h were concentrated 10-fold and then analyzed using liquid chromatography with iterative tandem mass spectrometry (LC-MS/MS). Using this approach on dialysate from the rat striatum with both reversed-phase and hydrophilic interaction liquid chromatography yielded 479 unique compound identifications. 60% of the identified compounds could be detected in 5 μL of dialysate without further concentration using a single 20 min LC-MS analysis, showing that once identified, most compounds can be detected using small sample volumes and shorter analysis times compatible with routine in vivo monitoring. To detect more neurochemicals, LC-MS analysis of dialysate derivatized with light and isotopically labeled benzoyl chloride was employed. 872 nondegenerate benzoylated features were detected with this approach, including most small-molecule neurotransmitters and several metabolites involved in dopamine metabolism. This strategy allows deeper annotation of the brain extracellular space than previously possible and provides a launching point for defining the chemistry underlying brain states.

Effect of Noopept on the Content of Neurotransmitter Amino Acids in the Hippocampus in Alcoholized Rats Using In Vivo Microdialysis

Effect of Noopept on the Content of Neurotransmitter Amino Acids in the Hippocampus in Alcoholized Rats Using In Vivo Microdialysis

The Effect of Aging on Nitric Oxide Production during Cerebral Ischemia and Reperfusion in Wistar Rats and Spontaneous Hypertensive Rats: An In Vivo Microdialysis Study.

Nitric oxide (NO) is involved in the pathogenesis of cerebral ischemic injury. Here, we investigated the effects of aging on NO production during cerebral ischemia-reperfusion (IR). Male Wister rats (WRs) were assigned to 12-month-old (older; n = 5) and 3-month-old (younger; n = 7) groups. Similarly, male spontaneous hypertensive rats (SHRs) were allocated to 12-month-old (older; n = 6) and 3-month-old (younger; n = 8) groups. After anesthesia, their NO production was monitored using in vivo microdialysis probes inserted into the left striatum and hippocampus. Forebrain cerebral IR injuries were produced via ligation of the bilateral common carotid arteries, followed by reperfusion. The change in the NO3- of the older rats in the SHR groups in the striatum was less compared to that of the younger rats before ischemia, during ischemia, and after reperfusion (p < 0.05). In the hippocampus, the change in the NO3- of the older rats in the SHR groups was lower compared to that of the younger rats after reperfusion (p < 0.05). There were no significant differences between the two WR groups. Our findings suggested that aging in SHRs affected NO production, especially in the striatum, before and during cerebral ischemia, and after reperfusion. Hypertension and aging may be important factors impacting NO production in brain IR injury.

Combined In Vivo Microdialysis and PET Studies to Validate [11C]Yohimbine Binding as a Marker of Noradrenaline Release.

The noradrenaline system attracts attention for its role in mood disorders and neurodegenerative diseases but the lack of well-validated methods impairs our understanding when assessing its function and release in vivo. This study combines simultaneous positron emission tomography (PET) and microdialysis to explore if [11C]yohimbine, a selective antagonist radioligand of the α2 adrenoceptors, may be used to assess in vivo changes in synaptic noradrenaline during acute pharmacological challenges. Anesthetised Göttingen minipigs were positioned in a head holder in a PET/CT device. Microdialysis probes were placed in the thalamus, striatum and cortex and dialysis samples were collected every 10 min. Three 90 min [11C]yohimbine scans were acquired: at baseline and at two timepoints after the administration of amphetamine (1-10 mg/kg), a non-specific releaser of dopamine and noradrenaline, or nisoxetine (1 mg/kg), a specific noradrenaline transporter inhibitor. [11C]yohimbine volumes of distribution (VT) were obtained using the Logan kinetic model. Both challenges induced a significant decrease in yohimbine VT, with time courses reflecting their different mechanisms of action. Dialysis samples revealed a significant increase in noradrenaline extracellular concentrations after challenge and an inverse correlation with changes in yohimbine VT. These data suggest that [11C]yohimbine can be used to evaluate acute variations in synaptic noradrenaline concentrations after pharmacological challenges.

Supplemental Material for In Vivo Microdialysis Shows Differential Effects of Prenatal Protein Malnutrition and Stress on Norepinephrine, Dopamine, and Serotonin Levels in Rat Orbital Frontal Cortex

Supplemental Material for In Vivo Microdialysis Shows Differential Effects of Prenatal Protein Malnutrition and Stress on Norepinephrine, Dopamine, and Serotonin Levels in Rat Orbital Frontal Cortex

Omadacycline Tissue Penetration in Diabetic Patients With Wound Infections and Healthy Volunteers Via In Vivo Microdialysis

Omadacycline Tissue Penetration in Diabetic Patients With Wound Infections and Healthy Volunteers Via In Vivo Microdialysis

Pathodynamics of Nitric Oxide Production Within Implanted Glioma Studied With an in Vivo Microdialysis Technique and Immunohistochemistry

Nitric oxide (NO) is thought to be a mediator in many of the processes of malignant brain tumor progression. We examined NO production in the brain of normal conscious, freely moving rats with or without implanted C6 glioma. Both nitrite (NO2-) and nitrate (NO3-) in the dialysates of the two groups were measured using an in vivo microdialysis technique. The mean concentration of NO2- in the glioma group was two-times higher than that in the control group (P<0.01). Concentrations of both NO2- and NO3- in the glioma and control groups decreased following intraperitoneal injection of NG-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NO synthase (NOS). NO production was also significantly suppressed in the glioma group, but not the control group, by intraperitoneal injection of 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), a selective inhibitor of inducible NOS (iNOS). On immunohistochemical examination, diffuse iNOS-positive cells were located within glioma tissue. ED1-positive cells (microglia/macrophages) were intermingled between glioma cells on double immunostaining. These results indicate that the basal level of NO production in the glioma group is higher than that in the control group and that the increased NO production was continuously induced by iNOS-expressing cells in glioma.

In Vivo Microdialysis Method to Collect Large Extracellular Proteins from Brain Interstitial Fluid with High-molecular Weight Cut-off Probes.

In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.

Use and Future Prospects of in Vivo Microdialysis for Epilepsy Studies.

Epilepsy is a common neurological disease characterized by recurrent unpredictable seizures. For the last 30 years, microdialysis sampling has been used to measure changes in excitatory and inhibitory neurotransmitter concentrations before, during, and after seizures. These advances have fostered breakthroughs in epilepsy research by identifying neurochemical changes associated with seizures and correlating them to electrophysiological data. Recent advances in methodology may be useful in further delineating the chemical underpinnings of seizures. A new model of ictogenesis has been developed that allows greater control over the timing of seizures that are similar to spontaneous seizures. This model will facilitate making chemical measurements before and during a seizure. Recent advancements in microdialysis sampling, including the use of segmented flow, "fast" liquid chromatography (LC), and capillary electrophoresis with laser-induced fluorescence (CE-LIF) have significantly improved temporal resolution to better than 1 min, which could be used to measure transient, spontaneous neurochemical changes associated with seizures. Microfabricated sampling probes that are markedly smaller than conventional probes and allow for a much greater spatial resolution have been developed. They may allow the targeting of specific brain regions important to epilepsy studies. Coupling microdialysis sampling to optogenetics and light-stimulated release of neurotransmitters may also prove useful for studying epileptic seizures.

Multiple Administration of Endogenous Amines TIQ and 1MeTIQ Protects Against a 6-OHDA-Induced Essential Fall of Dopamine Release in the Rat Striatum: In Vivo Microdialysis Study

Parkinson’s disease (PD) represents one of the neurodegenerative disorders which are caused by degeneration of dopaminergic neurons in the nigrostriatal pathway. Different toxins, e.g., 6-hydroxydopamine (6-OHDA), are used to model PD in animals. 6-OHDA is a neurotoxin which damages catecholaminergic neurons via production of oxygen radicals. Tetrahydroisoquinolines (TIQs) are endogenous amines which are present in the mammalian brain. Some of them, like TIQ and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ), demonstrate neuroprotective properties. These compounds act as reversible MAO inhibitors and this way block free radical formation. To continue our previous experiments, we evaluated the effect of acute and chronic treatment with TIQ and 1MeTIQ on locomotor/exploratory activity and the release of dopamine as well as its metabolite 3-methoxytyramine (3-MT) in the striatum of unilaterally 6-OHDA-lesioned and sham-operated rats using in vivo microdialysis methodology. Additionally, the changes in the concentration of tyrosine hydroxylase in the substantia nigra were measured. A unilateral 6-OHDA lesion in the substantia nigra produces a strong reduction in the release of dopamine (approx. 70%) and 3-MT (approx. 50%) in the rat striatum. This effect was completely inhibited by multiple administration of TIQ and 1MeTIQ. The results obtained from the in vivo microdialysis study suggest that multiple treatment with both endogenous amines, TIQ and 1MeTIQ, protects dopaminergic neurons against a 6-OHDA-induced deficit of dopamine release. Furthermore, these amines were able to maintain physiological functions of striatal dopamine neurons damaged by a unilateral 6-OHDA lesion.

Rational Design of a Stimuli-Responsive Polymer Electrode Interface Coupled with in Vivo Microdialysis for Measurement of Sialic Acid in Live Mouse Brain in Alzheimer's Disease.

Sensitive and selective monitoring of sialic acid (SA) in cerebral nervous system is of great importance for studying the role that SA plays in the pathological process of Alzheimer's disease (AD). In this work, we first reported an electrochemical biosensor based on a novel stimuli-responsive copolymer for selective and sensitive detection of SA in mouse brain. Notably, through synergetic hydrogen-bonding interactions, the copolymer could translate the recognition of SA into their conformational transition and wettability switch, which facilitated the access and enrichment of redox labels and targets to the electrode surface, thus significantly improving the detection sensitivity with the detection limit down to 0.4 pM. Besides amplified sensing signals, the proposed method exhibited good selectivity toward SA in comparison to potential interference molecules coexisting in the complex brain system due to the combination of high affinity between phenylboronic acid (PBA) and SA and the directional hydrogen-bonding interactions in the copolymer. The electrochemical biosensor with remarkable analytical performance was successfully applied to evaluate the dynamic change of SA level in live mouse brain with AD combined with in vivo midrodialysis. The accurate concentration of SA in different brain regions of live mouse with AD has been reported for the first time, which is beneficial for progressing our understanding of the role that SA plays in physiological and pathological events in the brain.

Monitoring Dopamine Responses to Potassium Ion and Nomifensine by in Vivo Microdialysis with Online Liquid Chromatography at One-Minute Resolution.

Recently, our laboratory has demonstrated the technical feasibility of monitoring dopamine at 1 min temporal resolution with microdialysis and online liquid chromatography. Here, we monitor dopamine in the rat striatum during local delivery of high potassium/low sodium or nomifensine in awake-behaving rats. Microdialysis probes were implanted and perfused continuously with or without dexamethasone in the perfusion fluid for 4 days. Dexamethasone is an anti-inflammatory agent that exhibits several positive effects on the apparent health of the brain tissue surrounding microdialysis probes. Dopamine was monitored 1 or 4 days after implantation under basal conditions, during 10 min applications of 60 mM or 100 mM K+, and during 15 min applications of 10 μM nomifensine. High K+ and nomifensine were delivered locally by adding them to the microdialysis perfusion fluid using a computer-controlled, low-dead-volume six-port valve. Each day/K+/dexamethasone combination elicited specific dopamine responses. Dexamethasone treatment increased dopamine levels in basal dialysates (i.e., in the absence of K+ or nomifensine). Applications of 60 mM K+ evoked distinct responses on days one and four after probe implantation, depending upon the presence or absence of dexamethasone, consistent with dexamethasone's ability to mitigate the traumatic effect of probe implantation. Applications of 100 mM K+ evoked dramatic oscillations in dopamine levels that correlated with changes in the field potential at a metal electrode implanted adjacent to the microdialysis probe. This combination of results indicates the role of spreading depolarization in response to 100 mM K+. With 1 min temporal resolution, we find that it is possible to characterize the pharmacokinetics of the response to the local delivery of nomifensine. Overall, the findings reported here confirm the benefits arising from the ability to monitor dopamine via microdialysis at high sensitivity and at high temporal resolution.

In Vivo Microdialysis of Brain Interstitial Fluid for the Determination of Extracellular Tau Levels.

Tau is a microtubule binding protein highly abundant in cytoplasm of neurons. However now there is compelling evidence that it is also physiologically released in the extracellular space. Tau is detectable in culture media of various cells, cerebrospinal fluid (CSF), and brain interstitial fluid (ISF). Being able to measure extracellular tau levels in vivo would deep our understanding on the underlying mechanisms of physiological/pathological tau release from neurons. To meet this need, this chapter describes an in vivo microdialysis protocol and materials for the measurement of extracellular tau in ISF from freely moving mice.

Identification of Lipid Mediators in Peripheral Human Tissues Using an Integrative In Vivo Microdialysis Approach

Endocannabinoids and related N-acylethanolamines (NAEs) are lipid mediators involved in a number of physiological and pathological mechanisms in peripheral tissues. Microdialysis (MD) technique all ...

Vancomycin Tissue Pharmacokinetics in Patients with Lower-Limb Infections via In Vivo Microdialysis.

Vancomycin is a common treatment option for skin and skin structure infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Given the increasing prevalence of MRSA, vancomycin is widely used as empirical therapy. In patients with lower-limb infections, antimicrobial penetration is often reduced because of decreased vascular perfusion. In this study, we evaluated the tissue concentrations of vancomycin in hospitalized patients with lower-limb infections. An in vivo microdialysis catheter was inserted near the margin of the wound and was perfused with lactated Ringer's solution. Tissue and serum samples were obtained after steady state for one dosing interval. Tissue concentrations were corrected for percentage of in vivo recovery using the retrodialysis technique. Nine patients were enrolled (mean ± SD: age, 54 ± 19 years; weight, 105.6 ± 31.5 kg). Patients received a mean of 12.8 mg/kg of vancomycin every 12 hours (n = 7), every 8 hours (n = 1), or every 24 hours (n = 1). Mean ± SD steady-state trough vancomycin concentrations in serum and tissue were 11.1 ± 3.3 and 6.0 ± 2.6 μg/mL. The mean ± SD 24-hour free drug areas under the curve for serum and wound were 283.7 ± 89.4 and 232.8 ± 75.7 μg*h/mL, respectively. The mean ± SD tissue penetration ratio was 0.8 ± 0.2. These data suggest that against MRSA with minimum inhibitory concentrations of 1 μg/mL or less, vancomycin achieved blood pharmacodynamic targets required for the likelihood of success. Reduced concentrations may contribute to poor outcomes and the development of resistance. As other literature suggests, alternative agents may be needed when the pathogen of interest has a minimum inhibitory concentration greater than 1 μg/mL.

Decline of Lactate in Tumor Tissue After Ketogenic Diet: In Vivo Microdialysis Study in Patients with Head and Neck Cancer

In head and neck squamous cell carcinoma (HNSCC) aerobic glycolysis is the key feature for energy supply of the tumor. Quantitative microdialysis (μD) offers an online method to measure parameters of the carbohydrate metabolism in vivo. The aim was to standardize a quantitative μD-study in patients with HNSCC and to prove if a ketogenic diet would differently influence the carbohydrate metabolism of the tumor tissue. Commercially available 100 kDa-CMA71-μD- catheters were implanted in tumor-free and in tumor tissue in patients with HNSCC for simultaneous measurements up to 5 days. The metabolic pattern and circadian rhythm of urea, glucose, lactate, and pyruvate was monitored during 24 h of western diet and subsequent up to 4 days of ketogenic diet. After 3 days of ketogenic diet the mean lactate concentration declines to a greater extent in the tumor tissue than in the tumor-free mucosa, whereas the mean glucose and pyruvate concentrations rise. The in vivo glucose metabolism of the tumor tissue is clearly influenced by nutrition. The decline of mean lactate concentration in the tumor tissue after ketogenic diet supports the hypothesis that HNSCC tumor cells might use lactate as fuel for oxidative glucose metabolism.

In Vivo Microdialysis Sampling of Cytokines from Rat Hippocampus: Comparison of Cannula Implantation Procedures

Cytokines are signaling proteins that have been of significant importance in the field of immunology, since these proteins affect different cells in the immune system. In addition to their immune system significance, these proteins have recently been referred to as a third chemical communication network within the CNS. The role that cytokines play in orchestrating the immune response within tissues after a mechanical injury leads to potential complications if the source of cytokines (i.e., trauma vs disease) is of interest. Microdialysis sampling has seen wide use in collection of many different solutes within the CNS. Yet, implantation of microdialysis guide cannulas and the probes creates tissue injury. In this study, we compared the differences in cytokine levels in dialysates from 4 mm, 100 kDa molecular weight cutoff (MWCO) polyethersulfone membrane microdialysis probes implanted in the hippocampus of male Sprague-Dawley rats. Comparisons were made between animals that were dialyzed immediately after cannula implantation (day 0), 7 days post cannula implantation (day 7), and repeatedly sampled on day 0 and day 7. Multiplexed bead-based immunoassays were used to quantify CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CXCL1 (KC/GRO), CXCL2 (MIP-2), IL-1β, IL-6, and IL-10 in dialysates. Differences in cytokine concentrations between the different treatment groups were observed with higher levels of inflammatory cytokines measured in day 7 cannulated animals. Only CCL3 (MIP-1α), CXCL1 (KC/GRO), CXCL2 (MIP-2), and IL-10 were measured above the assay limits of detection for a majority of the dialysates, and their concentrations were typically in the low to high (10-1000) picogram per milliliter range. The work described here lays the groundwork for additional basic research studies with microdialysis sampling of cytokines in rodent CNS.

Acute Effects of Pyrethroids on Serotonin Release in the Striatum of Awake Rats: An In Vivo Microdialysis Study

The present study examined the acute neurotoxic effects of three different pyrethroids, allethrin, cyhalothrin, and deltamethrin on the release of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the striatum of conscious rats using microdialysis. Allethrin 10 mg/kg reduced extracellular levels of 5-HT to 46%, whereas 20 and 60 mg/kg increased the release to 177% and 243% of baseline, respectively. Cyhalothrin increased 5-HT release to 145-204% and deltamethrin decreased to 58-32% of baseline in a dose-dependent manner. None of the pyrethroids tested altered extracellular levels of 5-HIAA. Local infusion of the voltage-gated sodium channel antagonist tetrodotoxin (TTX) into striatum completely prevented the effects of allethrin, cyhalothrin, and deltamethrin (10 and 20 mg/kg) on 5-HT release. The effect of deltamethrin at 60 mg/kg was completely abolished by striatal infusion of nimodipine (L-type Ca⁺⁺ channel antagonist) with TTX. These findings suggest that pyrethroids disrupt the serotonergic neurotransmission in striatum in a dose-related manner with Na⁺ and Ca²⁺ channel-dependent mechanisms.

Characterization of Basal and Morphine-Induced Uridine Release in the Striatum: An In Vivo Microdialysis Study in Mice

Uridine, a pyrimidine nucleoside, has been proposed to be a potential signaling molecule in the central nervous system. The understanding of uridine release in the brain is therefore of fundamental importance. The present study was performed to determine the characteristics of basal and morphine-induced uridine release in the striatum of freely moving mice by using the microdialysis technique. To ascertain whether extracellular uridine was derived from neuronal release, the following criteria were applied: sensitivity to (a) K(+) depolarization, (b) Na(+) channel blockade and (c) removal of extracellular Ca(2+). Uridine levels were not greatly affected by infusion of tetrodotoxin (TTX) and were unaffected by either Ca(2+)-free medium or in the presence of EGTA (a calcium chelator), suggesting that basal extracellular uridine levels were maintained mainly by non-vesicular release mechanisms. In addition, both systemic and local application of morphine increased striatal uridine release. The morphine-induced release was reversed by naloxone pretreatment, but was unaffected by TTX or EGTA infusion. Moreover, co-administration of morphine and nitrobenzylthioinosine (NBTI, an inhibitor of nucleotide transporter) produced increases of uridine levels similar to that produced by NBTI or morphine alone, suggesting a nucleotide transporter mechanism involved. Taken together, these findings suggest that morphine produces a μ-opioid receptor-mediated uridine release via nucleoside transporters in a TTX- and calcium-independent manner.

Differential Changes of Extracellular Aspartate and Glutamate in the Striatum of Domestic Chicken Evoked by High Potassium or Distress: An In Vivo Microdialysis Study

It has long been proposed that L: -aspartate (Asp) is an excitatory neurotransmitter similar to L: -glutamate (Glu) but with distinct signaling properties. The presence of Asp in excitatory synapses of the medial striatum/nucleus accumbens of domestic chicks suggests that Asp plays a role of neurotransmitter also in the avian brain. Neurotransmitters are released from the presynaptic bouton mostly by Ca(2+) dependent exocytosis. We used in vivo microdialysis to monitor the simultaneous changes of the extracellular levels of Asp and Glu in the medial striatum of young post-hatch domestic chicks. Microdialysis samples were collected from freely moving birds at 5 min intervals and analysed off-line using capillary electrophoresis. Event-related elevations of extracellular Glu and Asp concentrations in response to handling stress and to high KCl (50 mM) were observed. Increase of Glu and Asp on handling stress was 200 and 250 %, whereas on KCl stimulation the values were 300 and 1,000 %, respectively, if stress was applied before high KCl, and 150 and 200 %, respectively, in the absence of stress. In most cases, the amino acids showed correlated changes, Asp concentrations being consistently smaller at resting but exceeding Glu during stimulation. Using Ca(2+) free medium, the KCl triggered elevation of Glu was reduced. When KCl stimulation was combined with tetrodotoxin infusion, there was no significant elevation in Asp or in Glu suggesting that most of the extracellular excitatory amino acids were released by synaptic mechanisms. The results support the suggestion that Asp is co-released with Glu and may play a signaling role (as distinct from that of glutamate) in the striatum of birds.