Probe Recovery Research Articles

Unbound plasma concentrations may predict neuroprotective brain concentrations: a brain microdialysis and pharmacokinetic study of enadoline in rats.

A rat brain microdialysis study of enadoline (CI-977), a k-opioid agonist, was conducted in nonanesthetized healthy rats to determine brain extracellular fluid (ECF) concentrations of CI-977 associated with neuroprotective subcutaneous (s.c.) doses. Three groups of 3 to 4 nonanesthetized yet restrained Sprague-Dawley rats with jugular cannulas and implanted brain (striatum) microdialysis probes received single s.c. doses of 0.3, 1.0, or 3.0 mg/kg CI-977. Blood and microdialysate samples were collected over a 12-hour period. Extent of rat plasma protein binding was 77.5%. Unbound plasma concentrations associated with neuroprotection were 10-50 ng eq/mL. At each dose, brain ECF concentration-time profiles (corrected for probe recovery) were nearly coincident with enadoline plasma unbound concentration-time profiles. Consequently, at each dose the ratio of AUCecf/AUCffplasma, (AUC = Area Under the concentration-time Curve; ffplasma = free fraction in plasma = unbound plasma) which represents the distribution of drug between plasma and brain, was determined to be unity within experimental error. These results suggest that unbound plasma concentrations may predict brain ECF concentrations of CI-977. Further, our findings allow us to postulate enadoline unbound brain ECF concentrations necessary for neuroprotection.

Effects of hindlimb contraction on pressor and muscle interstitial metabolite responses in the cat.

We used the microdialysis technique to measure the interstitial concentration of several putative metabolic stimulants of the exercise pressor reflex during 3- and 5-Hz twitch contractions in the decerebrate cat. The peak increases in heart rate and mean arterial pressure during contraction were 20 +/- 5 beats/min and 21 +/- 8 mmHg and 27 +/- 9 beats/min and 37 +/- 12 mmHg for the 3- and 5-Hz stimulation protocols, respectively. All variables returned to baseline after 10 min of recovery. Interstitial lactate rose (P < 0. 05) by 0.41 +/- 0.15 and 0.56 +/- 0.16 mM for the 3- and 5-Hz stimulation protocols, respectively, and were not statistically different from one another. Interstitial lactate levels remained above (P < 0.05) baseline during recovery in the 5-Hz group. Dialysate phosphate concentrations (corrected for shifts in probe recovery) rose with stimulation (P < 0.05) by 0.19 +/- 0.08 and 0.11 +/- 0.03 mM for the 3- and 5-Hz protocols. There were no differences between groups. The resting dialysate K+ concentrations for the 3- and 5-Hz conditions were 4.0 +/- 0.1 and 3.9 +/- 0.1 meq/l, respectively. During stimulation the dialysate K+ concentrations rose steadily for both conditions, and the increase from rest to stimulation (P < 0.05) was 0.57 +/- 0.19 and 0.81 +/- 0.06 meq/l for the 3- and 5-Hz conditions, respectively, with no differences between groups. Resting dialysate pH was 6.915 +/- 0.055 and 6.981 +/- 0.032 and rose to 7.013 (P < 0.05) and 7.053 (P < 0.05) for the 3- and 5-Hz conditions, respectively, and then became acidotic (6. 905, P < 0.05) during recovery (5 Hz only). This study represents the first time simultaneous measurements of multiple skeletal muscle interstitial metabolites and pressor responses to twitch contractions have been made in the cat. These data suggest that interstitial K+ and phosphate, but not lactate and H+, may contribute to the stimulation of thin fiber muscle afferents during contraction.

Evaluation of microdialysis sampling of aqueous humor for in vivo models of ocular absorption and disposition

The dynamics of β-adrenergic-associated reductions in aqueous humor production for treatment of elevated intraocular pressure are not well understood. In particular, the relationship between ocular pharmacokinetics and pharmacodynamics has yet to be established. This study was undertaken to develop a procedure for examining the ocular absorption and disposition of topically administered ophthalmic β-adrenergic antagonists in individual animals. Dogs were anesthetized with isoflurane and a microdialysis probe was implanted in the anterior chamber of one eye and perfused with 0.9% saline at a rate of 2 μl min −1. 3H-propranolol was administered by intracameral injection or topically. Each dog received intracameral and topical propranolol, in alternate eyes on separate days, in a randomized cross-over fashion. Microdialysis probe effluent was collected every 5 min for ≥2.5 h; concentrations of propranolol were determined by liquid scintillation spectroscopy and were corrected for probe recovery of the substrate as determined by in vivo retrodialysis (∼46%) to estimate aqueous humor concentrations. In separate experiments in rabbits, microdialysis probes were implanted in each eye. 3H-propranolol was administered topically to one eye; the contralateral eye received intracameral 3H-propranolol. Model-independent pharmacokinetic parameters for each treatment phase were calculated. The mean±S.D. times to peak concentration of propranolol in aqueous humor were 86.6±47.6 min in the dog and 54.1±20.4 min in the rabbit. The terminal rate constant was 0.0189±0.00429 min −1 in the dog vs. 0.00983±0.00546 min −1 in the rabbit. Intraocular tissue availability of propranolol differed markedly between the dog ( n=3) and rabbit ( n=3) (∼0.056 in the dog vs. ∼0.55 in the rabbit). These results demonstrate the utility of microdialysis sampling for examination of ocular pharmacokinetics.

Radioimmunoassay for the measurement of S9788 in serum and microdialysis samples

S59788, 6-[4-(2,2-di-(fluorophenyl)-ethylaminol)-1-piperidinyl]- N, N′-di-2-propenyl-1,3,5-triazine-2,4-diamine, is a novel compound designed to reverse tumour multidrug resistance associated with cancer chemotherapy. A specific and sensitive radioimmunoassay has been developed for the analysis of S9788 in serum samples and adapted for samples obtained by microdialysis. The limit of quantitation is 0.2 ng ml −1 in perfusion medium and there is no cross reactivity of the antibody with known metabolites of the parent compound or with certain cytotoxic compounds likely to be coadministered with S9788. Maximum probe recovery during microdialysis was 66% at a flow of 1 μl min −1, using Ringer/BSA (70 mg ml −1) as the perfusion medium. The assay has sufficient sensitivity, precision, accuracy and specificity for the analysis of rat and human serum and microdialysis perfusate samples. The assay has been successfully applied to the determination of S9788 in rat plasma (total concentration) and the microdialysate of the same samples.

Is the sugar intestinal permeability test a reliable investigation for coeliac disease screening?

The lactulose/mannitol (L/M) intestinal permeability test is a simple, non-invasive screening test for coeliac disease. The reliability of the L/M test has so far only been tested in selected groups of patients with coeliac disease. To evaluate the reliability of the L/M test in a group of patients with coeliac disease who had been diagnosed during mass serological screening of the general population. Twenty nine patients with coeliac disease detected by screening and 54 age matched coeliac disease free controls aged 11-15 years underwent an L/M test with 5 g lactulose and 2 g mannitol in isotonic aqueous solution. Urinary sugars were measured by high performance liquid chromatography. The median % urinary recovery of lactulose (lactulose UR) was significantly higher in patients with coeliac disease than in controls (0.63 v 0.18, p < 0.001). The mean mannitol % UR was lower in patients with coeliac disease than in controls (17.6 v 18.5) but the difference was not significant. The median urinary L%/M% ratio was significantly higher in patients with coeliac disease than in controls (0.038 v 0.014, p < 0.001). However, 16 of the 29 patients with coeliac disease showed an L%/M% ratio within normal limits (< 0.044). The L/M intestinal permeability test is not a valuable tool for screening of coeliac disease in the general population. The pattern of the urinary probe recovery suggests that many patients with coeliac disease could remain symptomless because the extent of their intestinal mucosal damage is small ("short" coeliac disease).

Clinical microdialysis: the role of on-line measurement and quantitative microdialysis.

The use of microdialysis in the clinic is examined in the light of lessons learnt from microdialysis in freely moving rats. Changes in concentrations of metabolites are an important index of the state of health of tissues. For effective therapeutic intervention rapid assays are essential Enzyme-based on-line assays for glucose and lactate are described. By combining two of these assays simultaneous measurements of glucose and lactate, sampled at 2 min intervals can be obtained. The relation between dialysate concentrations and the true extracellular concentration of an analyte is dependent on conditions in the tissue sampled and cannot be calculated from in vitro probe recoveries. Furthermore, with acute implantation of the probe and possibly rapidly changing tissue conditions, there will be changes in probe recovery in vivo. Quantitative microdialysis allows the measurement of the true extracellular concentration and the probe recovery in vivo. The clinical applicability of a number of quantitative microdialysis methods is discussed, and three approaches highlighted. By increasing membrane length and reducing flow rate recovery in vivo can be increased to 100%. In this case dialysate concentrations equal extracellular ones. By perfusing an inert exogenous compound an index of changes to extracellular volume and hence tissue oedema can be obtained. In the zero net flux method the infusion of a few concentrations of the analyte under study allows the direct determination of both the ECF concentration and the in vivo recovery. The latter can provide valuable information about changes in the physical as well as chemical state of the tissue. This can guide rapid effective therapeutic intervention.

Galileo entry probe recovery of fine structure in the zonal wind profile at Jupiter

The algorithm developed to recover the zonal (east‐west) wind profile at Jupiter by Doppler tracking of the Galileo probe is well established in theory and verified by computer simulation. We extend the Doppler wind recovery algorithm to include detection and extraction of microscale wind structure. When the probe encounters regions of high wind shear, turbulence, atmospheric waves, or nonsteady aerodynamics (buffeting) a rapidly varying component is introduced into the probe telemetry Doppler data. These effects can be isolated by their respective Doppler signatures. Analysis of residuals allows fine structure in the wind profile to be located and retrieved. The overall wind recovery is a three‐step process: (1) The large‐scale zonal wind profile is recovered by converting the probe telemetry frequency residuals (as caused by the Doppler effect) to a velocity and back‐projecting it into the local horizontal east‐west direction at the probe location. The zonal winds are derived from the velocity residuals by a least squares algorithm. (2) Regions of high zonal wind shear are recovered numerically relative to the large‐scale wind structure and serve as a correction to the previously derived large‐scale profile. (3) The probe trajectory is updated to reflect both the large‐ and small‐scale wind structure, and the large‐scale wind profile is recalculated. The Galileo Doppler wind experiment described previously and the wind microstructure retrieval algorithm outlined here are the only reasonable means by which the vertical profile of the zonal winds at Jupiter can be directly sampled.

Glutamate release from the ovine fetal brain during maternal hemorrhage. A study using chronic in utero cerebral microdialysis.

Glutamate has been implicated in the pathophysiology of neuronal injury associated with cerebral hypoxia-ischemia. A model using chronic in utero microdialysis was developed to sample the extracellular space of the fetal brain. Using this model, we tested the hypothesis that glutamate efflux from the parasagittal parietal cortex of near-term fetuses would increase during maternal hemorrhage. Twelve near-term fetal sheep were instrumented with vascular catheters, and a microdialysis probe(s) was implanted into the parasagittal parietal cortex. After a 3-day recovery period, the animals were subjected to maternal hemorrhage until either the fetal pH was < 7.00 or the fetus died. The extracellular glutamate concentration in the collected dialysate was determined by high pressure liquid chromatography (HPLC). Maternal hemorrhage resulted in an 80-90% decrease in uterine blood flow, a decrease fetal po2, and a mixed metabolic and respiratory fetal acidosis. There were two groups of fetuses, survivors (n = 5) and nonsurvivors (n = 7). The nonsurvivor group showed a large increase (10-30-fold) in peak glutamate release (P = 0.0015). Survivors demonstrated a small (threefold) increase that was not statistically significant (P = 0.065), unless one animal with very low probe recovery was excluded (P = 0.0048). Extracellular glutamate release from the fetal brain can occur during maternal hemorrhage with fetal acidemia. The pathophysiologic role (if any) of glutamate release in the survivors remains to be elucidated. Our results are consistent with the hypothesis that in utero release of glutamate occurs during periods of fetal asphyxia. This experimental preparation of chronic fetal brain microdialysis can be used to monitor the brain extracellular concentration of any dialyzable substance in response to stress, including maternal hemorrhage.

Quantitative microdialysis under transient conditions

A microdialysis method is described which allows for the quantitative determination of extracellular analyte concentration under transient conditions. The method provides the extracellular concentration and the in vivo probe recovery as a function of time. The technique is based on the method for steady-state conditions but differs in the use of a between-group rather than a within-group design. Following cocaine and amphetamine administration, a significantly greater increase in extracellular DA was found than was estimated from the dialysate using conventional microdialysis methods. The discrepancy is due to microdialysis probe recovery decreasing concurrently with the increase observed in extracellular DA following cocaine and amphetamine injections. The method estimates the extracellular concentration independently of any changes in recovery.

Intestinal permeability in allergic rats: nerve involvement in antigen-induced changes

In vivo uptake of the probe 51Cr-labeled EDTA from the jejunum of egg albumin (EA)-sensitized rats was compared with controls at baseline and after intraluminal antigen challenge. Probe recovery in blood was 60-80% greater in sensitized animals during the baseline period, suggesting that sensitization resulted in increased intestinal permeability. Sensitized, but not control, rats demonstrated a 15-fold increase in 51Cr-EDTA uptake after intraluminal antigen; no change occurred with an unrelated protein. Macromolecular recovery was also enhanced in sensitized animals, since serum levels of immunoreactive EA were elevated 14-fold compared with controls. Antigen challenge was accompanied by biochemical (protease release) and morphological (reduced numbers) evidence of mast cell degranulation in sensitized rats. The neurotoxin tetrodotoxin (applied directly to ligated jejunal segments) inhibited EA-induced uptake of 51Cr-EDTA and antigen. In isolated jejunum from sensitized rats, tetrodotoxin reduced secretory responses to luminal, but not serosal, antigen. These results indicate that neural factors may influence the uptake of molecules from the gut lumen during intestinal anaphylaxis.

Co‐Administration of the D2 Antagonist Pimozide Inhibits Up‐Regulation of Dopamine Release and Uptake Induced by Repeated Cocaine

To investigate the hypothesis that the D2 dopamine (DA) receptor regulates DA uptake, as well as release, in the nucleus accumbens (N ACC), rats were pretreated for 10 days with either the selective D2 antagonist pimozide (1.0 mg/kg, i.p.) or vehicle, followed 3 h later by either cocaine (20 mg/kg, i.p.) or saline. On day 11, a microdialysis method was performed in which various DA concentrations (0, 10, and 20 nM DA) were perfused through the dialysis probe to characterize the diffusion of DA through tissue to and from the microdialysis probe (recovery). This diffusion of DA has been shown to be sensitive to changes in release and uptake. Pimozide pretreatment was shown to attenuate significantly a cocaine-induced increase in the in vivo recovery of DA (p < 0.01). The in vivo recovery for the vehicle/cocaine group was 47 +/- 4%, whereas the in vivo recovery for the pimozide/cocaine group was 31 +/- 3%. There was no difference between the pimozide/cocaine and control groups (pimozide/saline, 26 +/- 2%; vehicle/saline, 26 +/- 3%). In vitro probe calibrations indicated no significant difference in probe efficiencies between groups. These data suggest that the D2 receptor is capable of modulating uptake as well as release of DA in the N ACC of the rat.

The use of microdialysis for studying the regional effects of pharmacological manipulation on extracellular levels of amino acids - some methodological aspects

The purpose of this study was to examine and validate the use of microdialysis for sampling and pharmacologically manipulating extracellular amino acids in the brain. Repeated use of microdialysis probes in acute intracerebral experiments did not significantly alter the relative recovery in vitro for the amino acids quantitated (GABA, aspartate, glutamate, glycine, taurine, and alanine). Regional differences in basal levels of some of the amino acids were detected in dialysates collected from the dorsomedial hypothalamus, striatum, and frontal cortex. The percent in vitro recoveries for the amino acids from the probes used in the three regions were not significantly different suggesting that the regional differences in basal levels of amino acids were functionally derived and not a consequence of variations in probe recovery. Perfusion with nipecotic acid, an inhibitor of GABA uptake, resulted in selective elevations in extracellular GABA in the three regions studied. Conversely, perfusion with high-potassium, a depolarizing agent, resulted in significant elevations in not only extracellular GABA but also aspartate, glutamate, and taurine. Thus, microdialysis is a method which can be employed to assess and to pharmacologically manipulate extracellular amino acids in the rat brain.

Quantitation of proteins and internal antigen pools by a monoclonal sandwich radioimmune assay

We describe a monoclonal sandwich radioimmune assay (MSRA) that is capable of measuring 0.5 fmoles of protein antigens. The basis of the assay is the ability of most rabbit heteroantisera directed against protein antigens to precipitate labeled monoclonal Fab-antigen complexes. The assay offers the advantages of specificity, sensitivity, linearity and rapidity. The MSRA circumvents the need for preparation of either highly purified protein antigens for use in radioimmune inhibition assays or affinity purified antisera for sandwich immunoassays. Utilizing variations of MSRA protocols, we determined that 48% of FcR γ2b/γ1, 34% of an 90,000 M r glycoprotein identified by monoclonal antibody 2D2C, and 24% of an 82,000 M r glycoprotein identified by monoclonal antibody 2E2A were accessible to labeled Fab probes at the plasma membrane. The total amount of antigen per cell was determined by dividing the amount of antigen at the cell surface (determined from binding saturation assays) by the percentage of surface/total antigen. This value of total antigen agrees well with the total cellular antigen pool determined by MSRA after appropriate corrections for probe saturation and recovery of pre-formed Fab-antigen complexes were made.