Combined Microdialysis Research Articles

Imaging Acute Metabolic Changes in Patients with Mild Traumatic Brain Injury Using Hyperpolarized [1-13C]Pyruvate.

SummaryTraumatic brain injury (TBI) involves complex secondary injury processes following the primary injury. The secondary injury is often associated with rapid metabolic shifts and impaired brain function immediately after the initial tissue damage. Magnetic resonance spectroscopic imaging (MRSI) coupled with hyperpolarization of 13C-labeled substrates provides a unique opportunity to map the metabolic changes in the brain after traumatic injury in real-time without invasive procedures. In this report, we investigated two patients with acute mild TBI (Glasgow coma scale 15) but no anatomical brain injury or hemorrhage. Patients were imaged with hyperpolarized [1-13C]pyruvate MRSI 1 or 6 days after head trauma. Both patients showed significantly reduced bicarbonate (HCO3–) production, and one showed hyperintense lactate production at the injured sites. This study reports the feasibility of imaging altered metabolism using hyperpolarized pyruvate in patients with TBI, demonstrating the translatability and sensitivity of the technology to cerebral metabolic changes after mild TBI.

PBPK model of methotrexate in cerebrospinal fluid ventricles using a combined microdialysis and MRI acquisition

The objective of the study was to evaluate the distribution of methotrexate (MTX) in cerebrospinal fluid (CSF) lateral ventricles and in cisterna magna after 3rd intraventricular CSF administration in a rabbit model.MTX or gadolinium chelate (Gd-DOTA) was administered in the 3rd ventricle with a local microdialysis to study the pharmacokinetics at the site of administration and with a simultaneous magnetic resonance imaging (MRI) acquisition in the 3rd ventricle, the lateral ventricles and in the cisterna magna. A specific CSF Physiologically Based Pharmacokinetic (PBPK) model was then extrapolated for MTX from Gd-DOTA data.The relative contribution of elimination and distribution processes to the overall disposition of MTX and Gd-DOTA in the 3rd ventricle was similar (i.e., around 60% for CLE and 40% for CLI) suggesting that Gd-DOTA was a suitable surrogate marker for MTX disposition in ventricular CSF. The PBPK predictions for MTX both in CSF of the 3rd ventricle and in plasma were in accordance with the in vivo results.The present study showed that the combination of local CSF microdialysis with MRI acquisition of the brain ventricles and a PBPK model could be a useful methodology to estimate the drug diffusion within CSF ventricles after direct brain CSF administration. Such a methodology would be of interest to clinicians for a rationale determination and optimization of drug dosing parameters in the treatment of leptomeningeal metastases.

Oxymorphone Active Uptake at the Blood–Brain Barrier and Population Modeling of its Pharmacokinetic–Pharmacodynamic Relationship

The aim of this study was to characterize the blood-brain barrier (BBB) transport and pharmacokinetics-pharmacodynamics (PKPD) relationship of oxymorphone and to further elucidate its possible contribution to oxycodone analgesia. The BBB transport of oxymorphone was studied using microdialysis in male Sprague-Dawley rats. Samples from microdialysis blood and brain probes, brain tissue, and plasma were analyzed by liquid chromatography with tandem mass spectrometry. The effect was measured as tail-flick latency. The study consisted of a PKPD experiment with combined microdialysis and antinociceptive measurements (n = 8), and another antinociceptive effect experiment (n = 9) using a 10 times lower dose. The combined data were analyzed with an integrated PKPD model in nonlinear mixed effect modeling utilizing a specific method (M3) for handling missing PD observations. The concentration of unbound oxymorphone was higher in brain than in blood, with a ratio of 1.9 (RSE, 9.7%), indicating active uptake at the BBB. The integrated PKPD model described the oxymorphone BBB transport and PKPD relationship successfully, with an EC50 in the brain of 63 ng/mL, and the M3 method was able to address the issue of censored observations. Oxymorphone has active uptake transport at the BBB in rats, with moderate uptake clearance to the brain. Its contribution to analgesia after oxycodone administration is not significant.

Immobilized Ru(bpy) 3 2�� Electrochemiluminescence Coupled with Microdialysis Sampling for On-line Studying Drug-Protein Interaction

An electrochemiluminescence(ECL) sensor based on the use of Nepem-105D perfluorinated ion exchange solution to form the ion exchange membrane on the surface of platinum electrode for immobilization of Ru(bpy)32+ has been developed.A new method was designed combined microdialysis sampling technology and ECL sensor for on-line study of the interactions between ciprofloxacin and bovine serum albumin(BSA).Under the optimized experimental conditions the relative ECL intensity is proportional with ciprofloxacin concentration in the range of 3.0×10-8-3.0×10-5 mol/L with a detection limit of 1.0×10-8 mol/L.The association constant of compound of ciprofloxacin and BSA and the number of the binding sites with Scatchard analysis are 3.51×104 L/mol and 1.49,respectively.The results show that the novel method can provide a fast and simple technique for the study of drug-protein interaction.

Endogenous nociceptin/orphanin FQ (N/OFQ) contributes to haloperidol-induced changes of nigral amino acid transmission and parkinsonism: a combined microdialysis and behavioral study in naïve and nociceptin/orphanin FQ receptor knockout mice

The contribution of endogenous nociceptin/orphanin FQ (N/OFQ) to neuroleptic-induced parkinsonism has been evaluated in haloperidol-treated mice. Pharmacological blockade of N/OFQ receptors (NOP) via systemic administration of 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397, 0.01–10 mg/kg i.p.) or central injection of [Nphe 1,Arg 14,Lys 15]N/OFQ-NH 2 (UFP-101, 10 nmol i.c.v.) attenuated (0.8 mg/kg) haloperidol-induced motor deficits as evaluated by a battery of behavioral tests providing complementary information on motor parameters: the bar, drag and rotarod tests. A combined neurochemical and behavioral approach was then used to investigate whether the substantia nigra reticulata could be involved in antiakinetic actions of J-113397. Microdialysis combined to the bar test revealed that haloperidol (0.3 and 0.8 mg/kg i.p.) caused a dose-dependent and prolonged elevation of immobility time (i.e. akinesia) which was associated with an increase in nigral glutamate and a reduction in GABA release. Conversely, J-113397 (1 mg/kg) alone reduced glutamate and elevated nigral GABA release, and when challenged against haloperidol, counteracted its behavioral and neurochemical effects. Microdialysis coupled to behavioral testing also demonstrated that NOP receptor knockout mice were resistant to haloperidol (0.3 mg/kg) compared to wild-type mice, lack of response being associated with a reversal of glutamate release facilitation into inhibition and no change in nigral GABA release. This study provides pharmacological and genetic evidence that endogenous N/OFQ contributes to haloperidol-induced akinesia and changes of amino acid transmission in mice. Moreover, it confirms the view that NOP receptor antagonists are capable of reversing akinesia across species and genotypes and may prove effective in relieving neuroleptic-induced parkinsonism.

A combined microdialysis and FDG-PET study of glucose metabolism in head injury

Microdialysis continuously monitors the chemistry of a small focal volume of the cerebral extracellular space. Positron emission tomography (PET) establishes metabolism of the whole brain but only for the scan's duration. This study's objective was to apply these techniques together, in patients with traumatic brain injury, to assess the relationship between microdialysis (extracellular glucose, lactate, pyruvate, and the lactate/pyruvate (L/P) ratio as a marker of anaerobic metabolism) and PET parameters of glucose metabolism using the glucose analogue [(18)F]-fluorodeoxyglucose (FDG). In particular, we aimed to determine the fate of glucose in terms of differential metabolism to pyruvate and lactate. Microdialysis catheters (CMA70 or CMA71) were inserted into the cerebral cortex of 17 patients with major head injury. Microdialysis was performed during FDG-PET scans with regions of interest for PET analysis defined by the location of the gold-tipped microdialysis catheter. Microdialysate analysis was performed on a CMA600 analyser. There was significant linear relationship between the PET-derived parameter of glucose metabolism (regional cerebral metabolic rate of glucose; CMRglc) and levels of lactate (r = 0.778, p < 0.0001) and pyruvate (r = 0.799, p < 0.0001), but not with the L/P ratio. The results suggest that in this population of patients, glucose was metabolised to both lactate and pyruvate, but was not associated with an increase in the L/P ratio. This suggests an increase in glucose metabolism to both lactate and pyruvate, as opposed to a shift towards anaerobic metabolism.

In vitro and in vivo protein sampling by combined microdialysis and ultrafiltration

Cytokines, chemokines and growth factors regulate inflammation, resistance to infection and tissue repair. Understanding their function within tissues is a priority in evolving therapy for a number of disease processes. Yet, the existence of complex networks of these factors in the tissue microenvironment has made understanding of their interactions difficult. We demonstrate the capability of microdialysis probes to recover small proteins efficiently in vitro. Further we show that microdialysis of human tissues allows for protein recovery from tissue interstitial fluid. This technology, combined with a multiplexed immunoassay, facilitates the simultaneous measurement of cytokines and chemokines in response to injury in the oral mucosa of human subjects in vivo.

Anaphylactic Shock

The pathophysiology of anaphylactic shock during anesthesia is incompletely characterized. It is described as distributive by analogy with septic shock (anaerobic metabolism, high tissue oxygen pressure [Ptio2] values). The Ptio2 profile and its metabolic consequences during anaphylaxis are not known. Ovalbumin-sensitized anaphylactic shock rats (n = 11) were compared to nicardipine-induced hypotension rats (n = 12) for systemic hemodynamics, Ptio2, sympathetic nervous system activation, skeletal muscle blood flow, and interstitial lactate and pyruvate concentrations using combined microdialysis and polarographic Clark-type oxygen probes. In both groups, the time course and the magnitude of arterial hypotension were similar. The ovalbumin group but not the nicardipine group displayed decreased skeletal muscle blood flow (from 45 +/- 6.2 ml x 100 g(-1) x min(-1) to 24.3 +/- 5 ml x 100 g(-1) x min(-1); P < 0.0001) and Ptio2 values (from 42 +/- 5 to 5 +/- 2; P < 0.0001). The ovalbumin group had more intense sympathetic nervous system activation with higher plasma epinephrine and interstitial norepinephrine concentrations. For the ovalbumin group, there was skeletal muscle anaerobic metabolism (lactate concentration increased from 0.446 +/- 0.105 to 1.741 +/- 0.459 mm; P < 0.05) and substrate depletion (pyruvate concentration decreased from 0.034 +/- 0.01 mm to 0.006 +/- 0.002 mm; P < 0.05) leading to increased interstitial lactate/pyruvate ratios (from 17 +/- 6 to 311 +/- 115; P < 0.05). This profile suggests decreased skeletal muscle blood flow and oxygen delivery. Persistent energy consumption results in decreased Ptio2 and substrate depletion through anaerobic glycolysis leading to complete failure of cellular energy production. This could explain rapid organ dysfunction and resuscitation difficulties.

Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study.

The nonspecific P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18-h food-deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding-induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 microm) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding-induced DA release and prevented the feeding-elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0-13.0-Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8-SPT (100 microm). When given alone, 8-SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding-elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8-SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding-induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding-associated mesolimbic neuronal activity in a functionally antagonistic manner.

Correlation between cerebral blood flow, substrate delivery, and metabolism in head injury: a combined microdialysis and triple oxygen positron emission tomography study.

Microdialysis continuously monitors the chemistry of a small focal volume of the cerebral extracellular space. Conversely, positron emission tomography (PET) establishes metabolism of the whole brain, but only for the duration of the scan. The objective of this study was to apply both techniques to head-injured patients simultaneously to assess the relation between microdialysis (glucose, lactate, lactate/pyruvate [L/P] ratio, and glutamate) and PET (cerebral blood flow [CBF], cerebral blood volume, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen) parameters. Microdialysis catheters were inserted into the frontal cerebral cortex and adipose tissue of the anterior abdominal wall of 17 severely head-injured patients. Microdialysis was performed during PET scans, with regions of interest defined by the location of the microdialysis catheter membrane. An intervention (hyperventilation) was performed in 13 patients. The results showed that combining PET and microdialysis to monitor metabolism in ventilated patients is feasible and safe, although logistically complex. There was a significant relation between the L/P ratio and the OEF (Spearman r = 0.69, P = 0.002). There was no significant relation between CBF and the microdialysis parameters. Moderate short-term hyperventilation appeared to be tolerated in terms of brain chemistry, although no areas were sampled by microdialysis where the OEF exceeded 70%. Hyperventilation causing a reduction of the arterial carbon dioxide tension by 0.9 kPa resulted in a significant elevation of the OEF, in association with a reduction in glucose, but no significant elevation in the L/P ratio or glutamate.

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min−1. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×104 M−1 and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.

Release of vasopressin from supraoptic neurons within the median eminence in vivo. A combined microdialysis and push-pull perfusion study in the rat

The present study was designed to investigate whether or not arginine vasopressin (AVP) is released from magnocellular neurons within the median eminence (ME) in vivo. Urethane-anesthetized adult male Wistar rats were equipped with a microdialysis probe aimed at the supraoptic (SON) or paraventricular nucleus (PVN), a push-pull perfusion probe resting in the ME, and a blood microdialysis probe within the jugular vein. Dialysis of the SON (but not the PVN) with Ringer's solution containing 56 mmol l −1 K + resulted in an increase in AVP release within the ME (to 492 ± 192% of release during basal conditions, P < 0.05) and into blood (to 138 ± 9%, P < 0.01) whereby the release probably occurred from axonal swellings and nerve terminals of supraoptic neurons which project through the internal zone of the ME to the posterior pituitary. The calculated amount of AVP released into the extracellular fluid of the ME was high enough (approximately 1 pg/μ1) to hypothesize that the neuropeptide could enter the portal blood capillaries in physiologically relevant concentrations. Taken together, the present study indicates that activation of magnocellular neurons is accompanied by release of AVP within the median eminence. We assume that AVP released in this way might mediate a communication between the hypothalamic-neurohypophysial system and the hypothalamic-pituitary-adrenal axis in response to selected stressful stimuli.

Release of vasopressin from supraoptic neurons within the median eminence in vivo. A combined microdialysis and push-pull perfusion study in the rat

The present study was designed to investigate whether or not arginine vasopressin (AVP) is released from magnocellular neurons within the median eminence (ME) in vivo. Urethane-anesthetized adult male Wistar rats were equipped with a microdialysis probe aimed at the supraoptic (SON) or paraventricular nucleus (PVN), a push-pull perfusion probe resting in the ME, and a blood microdialysis probe within the jugular vein. Dialysis of the SON (but not the PVN) with Ringer's solution containing 56 mmol l−1 K+ resulted in an increase in AVP release within the ME (to 492 ± 192% of release during basal conditions,P < 0.05) and into blood (to 138 ± 9%,P < 0.01) whereby the release probably occurred from axonal swellings and nerve terminals of supraoptic neurons which project through the internal zone of the ME to the posterior pituitary. The calculated amount of AVP released into the extracellular fluid of the ME was high enough (approximately 1 pg/μ1) to hypothesize that the neuropeptide could enter the portal blood capillaries in physiologically relevant concentrations. Taken together, the present study indicates that activation of magnocellular neurons is accompanied by release of AVP within the median eminence. We assume that AVP released in this way might mediate a communication between the hypothalamic-neurohypophysial system and the hypothalamic-pituitary-adrenal axis in response to selected stressful stimuli.

Direct detection of ascorbyl radical in experimental brain injury: microdialysis and an electron spin resonance spectroscopic study.

To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H2O2 and FeCl2 adjacent to the dialysis probe. When the rats were challenged with cold-induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl2 dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.

Simultaneous monitoring of intracerebral release and behavior: endogenous vasopressin improves social recognition.

We previously reported that direct osmotic stimulation of the supraoptic nucleus (SON) of rats via a microdialysis probe produces a robust 'rebound' release of endogenous vasopressin (AVP) into the extracellular fluid of this hypothalamic nucleus. We now demonstrate in a combined microdialysis and push-pull perfusion study that this intranuclear release is accompanied by increased AVP release in the septum. Simultaneous monitoring of intranuclear release and behavioral performance in the same animal indicated a significant correlation between the amount of endogenously released AVP and improved social memory based on the olfactory discriminative capacities of adult male rats. This memory improvement was partially blocked by local administration of a AVP V1 receptor antagonist either into the SON or septum. These findings indicate that direct osmotic stimulation of the supraoptic nucleus, which increases intracerebral vasopressin release, improves the acquisition and/or processing of olfactory stimuli. Thus, the endogenous neuropeptide fulfills one of the major criteria for being causally involved as a neurotransmitter/neuromodulator in behavioral performance.

Competitive inhibition of nitric oxide synthase prevents the cortical hyperemia associated with peripheral nerve stimulation.

With combined microdialysis and hydrogen clearance techniques for simultaneous local delivery of drugs and blood-flow measurement in the rat hindlimb sensory-motor cortex, we examined the role of nitric oxide in cerebral blood-flow regulation during sciatic nerve stimulation. Infusion of 1 mM nitric oxide synthase antagonist, N eta-nitro-L-arginine methyl ester (L-NAME), blocked the cortical blood-flow response to sciatic nerve stimulation (152 +/- 43 ml.min-1.100 g-1 of tissue in controls and 73 +/- 11 ml.min-1.100 g-1 in the presence of L-NAME; P less than 0.05). Addition of 10 mM L-arginine to the dialysate containing L-NAME partially restored the hyperemic response to nerve stimulation (125 ml.min-1.100 g-1). L-NAME also produced a decrease in baseline cerebral blood flow when compared with the control (66 +/- 14 ml.min-1.100 g-1 vs. 93 +/- 25 ml.min-1.100 g-1). We conclude that nitric oxide from activated neurons participates in the local regulation of cortical blood flow in response to sciatic nerve stimulation and also in the maintenance of basal cortical blood flow.