Deficits in basal and evoked striatal dopamine release following alpha-synuclein preformed fibril injection: An in vivo microdialysis study.

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.

Dual DAT and sigma receptor inhibitors attenuate cocaine effects on nucleus accumbens dopamine dynamics in rats.

Psychostimulant use disorders (PSUD) are prevalent; however, no FDA-approved medications have been made available for treatment. Previous studies have shown that dual inhibitors of the dopamine transporter (DAT) and sigma receptors significantly reduce the behavioral/reinforcing effects of cocaine, which have been associated with stimulation of extracellular dopamine (DA) levels resulting from DAT inhibition. Here, we employ microdialysis and fast scan cyclic voltammetry (FSCV) procedures to investigate the effects of dual inhibitors of DAT and sigma receptors in combination with cocaine on nucleus accumbens shell (NAS) DA dynamics in naïve male Sprague Dawley rats. In microdialysis studies, administration of rimcazole (3, 10 mg/kg; i.p.) or its structural analog SH 3-24 (1, 3 mg/kg; i.p.), compounds that are dual inhibitors of DAT and sigma receptors, significantly reduced NAS DA efflux stimulated by increasing doses of cocaine (0.1, 0.3, 1.0 mg/kg; i.v.). Using the same experimental conditions, in FSCV tests, we show that rimcazole pretreatments attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Under the same conditions, JJC8-091, a modafinil analog and dual inhibitor of DAT and sigma receptors, similarly attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Our results provide the neurochemical groundwork towards understanding actions of dual inhibitors of DAT and sigma receptors on DA dynamics that likely mediate the behavioral effects of psychostimulants like cocaine.

Continuous evaluation of single-dose moxifloxacin concentrations in brain extracellular fluid, cerebrospinal fluid, and plasma: a novel porcine model.

BACKGROUND: Knowledge regarding CNS pharmacokinetics of moxifloxacin is limited, with unknown consequences for patients with meningitis caused by bacteria resistant to beta-lactams or caused by TB. OBJECTIVE: (i) To develop a novel porcine model for continuous investigation of moxifloxacin concentrations within brain extracellular fluid (ECF), CSF and plasma using microdialysis, and (ii) to compare these findings to the pharmacokinetic/pharmacodynamic (PK/PD) target against TB. METHODS: Six female pigs received an intravenous single dose of moxifloxacin (6 mg/kg) similar to the current oral treatment against TB. Subsequently, moxifloxacin concentrations were determined by microdialysis within five compartments: brain ECF (cortical and subcortical) and CSF (ventricular, cisternal and lumbar) for the following 8 hours. Data were compared to simultaneously obtained plasma samples. Chemical analysis was performed by high pressure liquid chromatography with mass spectrometry. The applied PK/PD target was defined as a maximum drug concentration (Cmax):MIC ratio >8. RESULTS: We present a novel porcine model for continuous in vivo CNS pharmacokinetics for moxifloxacin. Cmax and AUC0-8h within brain ECF were significantly lower compared to plasma and lumbar CSF, but insignificantly different compared to ventricular and cisternal CSF. Unbound Cmax:MIC ratio across all investigated compartments ranged from 1.9 to 4.3. CONCLUSION: A single dose of weight-adjusted moxifloxacin administered intravenously did not achieve adequate target site concentrations within the uninflamed porcine brain ECF and CSF to reach the applied TB CNS target.

Characterization of the Neurochemical and Behavioral Effects of the Phenethylamine 2-Cl-4,5-MDMA in Adolescent and Adult Male Rats.

BACKGROUND: The proliferation of novel psychoactive substances (NPS) in the drug market raises concerns about uncertainty on their pharmacological profile and the health hazard linked to their use. Within the category of synthetic stimulant NPS, the phenethylamine 2-Cl-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA) has been linked to severe intoxication requiring hospitalization. Thereby, the characterization of its pharmacological profile is urgently warranted. METHODS: By in vivo brain microdialysis in adolescent and adult male rats we investigated the effects of 2-Cl-4,5-MDMA on dopamine (DA) and serotonin (5-HT) neurotransmission in two brain areas critical for the motivational and rewarding properties of drugs, the nucleus accumbens (NAc) shell and the medial prefrontal cortex (mPFC). Moreover, we evaluated the locomotor and stereotyped activity induced by 2-Cl-4,5-MDMA and the emission of 50-kHz ultrasonic vocalizations (USVs) to characterize its affective properties. RESULTS: 2-Cl-4,5-MDMA increased dialysate DA and 5-HT in a dose-, brain area-, and age-dependent manner. Notably, 2-Cl-4,5-MDMA more markedly increased dialysate DA in the NAc shell and mPFC of adult than adolescent rats, while the opposite was observed on dialysate 5-HT in the NAc shell, with adolescent rats being more responsive. Furthermore, 2-Cl-4,5-MDMA stimulated locomotion and stereotyped activity in both adolescent and adult rats, although to a greater extent in adolescents. Finally, 2-Cl-4,5-MDMA did not stimulate the emission of 50-kHz USVs. CONCLUSIONS: This is the first pharmacological characterization of 2-Cl-4,5-MDMA demonstrating that its neurochemical and behavioral effects may differ between adolescence and adulthood. These preclinical data could help understanding the central effects of 2-Cl-4,5-MDMA by increasing awareness on possible health damage in users.

Negligible In Vitro Recovery of Macromolecules from Microdialysis Using 100 kDa Probes and Dextran in Perfusion Fluid.

Microdialysis is applied in neurointensive care to monitor cerebral glucose metabolism. If recoverable, macromolecules may also serve as biomarkers in brain disease and provide clues to their passage across the blood-brain barrier. Our study aimed to investigate the in vitro recovery of human micro- and macromolecules using microdialysis catheters and perfusion fluids approved for clinical use. In vitro microdialysis of a bulk solution containing physiological or supraphysiological concentrations of glucose, lactate, pyruvate, human IgG, serum albumin, and hemoglobin was performed using two different catheters and perfusion fluids. One had a membrane cut-off of 20 kDa and was used with a standard CNS perfusion fluid, and the other had a membrane cut-off of 100 kDa and was perfused with the same solution supplemented with dextran. The flow rate was 0.3 µl/min. We used both push and push-pull methods. Dialysate samples were collected at 2-h intervals for 6 h and analyzed for relative recovery of each substance. The mean relative recovery of glucose, pyruvate, and lactate was > 90% in all but two sets of experiments. In contrast, the relative recovery of human IgG, serum albumin, and hemoglobin from both bulk solutions was below the lower limit of quantification (LLOQ). Using a push-pull method, recovery of human IgG, serum albumin, and hemoglobin from a bulk solution with supraphysiological concentrations were above LLOQ but with low relative recovery (range 0.9%-1.6%). In summary, exchanging the microdialysis setup from a 20 kDa catheter with a standard perfusion fluid for a 100 kDa catheter with a perfusion solution containing dextran did not affect the relative recovery of glucose and its metabolites. However, it did not result in any useful recovery of the investigated macromolecules at physiological levels, either with or without a push-pull pump system.

Microdialysis coupled with droplet microfluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.

Monitoring the concentration fluctuations of neurotransmitters in vivo is valuable for elucidating the chemical signals that underlie brain functions. Microdialysis sampling is a widely used tool for monitoring neurochemicals in vivo. The volume requirements of most techniques that have been coupled to microdialysis, such as HPLC, result in fraction collection times of minutes, thus limiting the temporal resolution possible. Further the time of analysis can become long for cases where many fractions are collected. Previously we have used direct analysis of dialysate by low-flow electrospray ionization-tandem mass spectrometry (ESI-MS/MS) on a triple quadrupole mass spectrometer to monitor acetylcholine, glutamate, and γ-amino-butyric acid to achieve multiplexed in vivo monitoring with temporal resolution of seconds. Here, we have expanded this approach to adenosine, dopamine, and serotonin. The method achieved limits of detection down to 2 nM, enabling basal concentrations of all these compounds, except serotonin, to be measured in vivo. Comparative analysis with LC-MS/MS showed accurate results for all compounds except for glutamate, possibly due to interference for this compound in vivo. Pairing this analysis with droplet microfluidics yields 11 s temporal resolution and can generate dialysate fractions down to 3 nL at rates up to 3 fractions per s from a microdialysis probe. The system is applied to multiplexed monitoring of neurotransmitter dynamics in response to stimulation by 100 mM K+ and amphetamine. These applications demonstrate the suitability of the droplet ESI-MS/MS method for monitoring short-term dynamics of up to six neurotransmitters simultaneously.

Understanding the nebulisation of antibiotics: the key role of lung microdialysis studies.

BACKGROUND: Nebulisation of antibiotics is a promising treatment for ventilator-associated pneumonia (VAP) caused by multidrug-resistant organisms. Ensuring effective antibiotic concentrations at the site of infection in the interstitial space fluid is crucial for clinical outcomes. Current assessment methods, such as epithelial lining fluid and tissue homogenates, have limitations in providing longitudinal pharmacokinetic data. MAIN BODY: Lung microdialysis, an invasive research technique predominantly used in animals, involves inserting probes into lung parenchyma to measure antibiotic concentrations in interstitial space fluid. Lung microdialysis offers unique advantages, such as continuous sampling, regional assessment of antibiotic lung concentrations and avoidance of bronchial contamination. However, it also has inherent limitations including the cost of probes and assay development, the need for probe calibration and limited applicability to certain antibiotics. As a research tool in VAP, lung microdialysis necessitates specialist techniques and resource-intensive experimental designs involving large animals undergoing prolonged mechanical ventilation. However, its potential impact on advancing our understanding of nebulised antibiotics for VAP is substantial. The technique may enable the investigation of various factors influencing antibiotic lung pharmacokinetics, including drug types, delivery devices, ventilator settings, interfaces and disease conditions. Combining in vivo pharmacokinetics with in vitro pharmacodynamic simulations can become feasible, providing insights to inform nebulised antibiotic dose optimisation regimens. Specifically, it may aid in understanding and optimising the nebulisation of polymyxins, effective against multidrug-resistant Gram-negative bacteria. Furthermore, lung microdialysis holds promise in exploring novel nebulisation therapies, including repurposed antibiotic formulations, bacteriophages and immunomodulators. The technique's potential to monitor dynamic biochemical changes in pneumonia, such as cytokines, metabolites and inflammation/infection markers, opens avenues for developing theranostic tools tailored to critically ill patients with VAP. CONCLUSION: In summary, lung microdialysis can be a potential transformative tool, offering real-time insights into nebulised antibiotic pharmacokinetics. Its potential to inform optimal dosing regimen development based on precise target site concentrations and contribute to development of theranostic tools positions it as key player in advancing treatment strategies for VAP caused by multidrug-resistant organisms. The establishment of international research networks, exemplified by LUMINA (lung microdialysis applied to nebulised antibiotics), signifies a proactive step towards addressing complexities and promoting multicentre experimental studies in the future.

Dermal microdialysis: A method to determine drug levels in the skin of patients with Post kala-azar dermal leishmaniasis (PKDL).

OBJECTIVES: Post-kala-azar-dermal leishmaniasis (PKDL) is an infectious skin disease that occurs as sequela of visceral leishmaniasis (VL) and causes cutaneous lesions on the face and other exposed body parts. While the first-line drug miltefosine is typically used for 28 days to treat VL, 12 weeks of therapy is required for PKDL, highlighting the need to evaluate the extent of drug penetration at the dermal site of infection. In this proof-of-concept study, we demonstrate the use of a minimally invasive sampling technique called microdialysis to measure dermal drug exposure in a PKDL patient, providing a tool for the optimization of treatment regimens. METHODS AND MATERIALS: One PKDL patient receiving treatment with miltefosine (50 mg twice daily for 12 weeks) was recruited to this proof-of-concept study and consented to undergo dermal microdialysis. Briefly, a µDialysis Linear Catheter 66 for skin and muscle, a probe with a semi-permeable membrane, was inserted in the dermis. A perfusate (a drug-free physiological solution) was pumped through the probe at a low flow rate, allowing miltefosine present in the dermis to cross the membrane and be collected in the dialysates over time. Protein-free (dialysates) and total (blood and skin biopsies) drug concentrations were analysed using LC-MS/MS. RESULTS: and conclusions: Using microdialysis, protein-free miltefosine drug concentrations could be detected in the infected dermis over time (Cmax ≈ 450 ng/ml). This clinical proof-of-concept study thus illustrates the potential of dermal microdialysis as a minimally invasive alternative to invasive skin biopsies to quantify drug concentrations directly at the pharmacological site of action in PKDL.

The effects of cerebral pressure autoregulation status and CPP levels on cerebral metabolism in pediatric traumatic brain injury.

BACKGROUND: Cerebral perfusion pressure (CPP) management in the developing child with traumatic brain injury (TBI) is challenging. The pressure reactivity index (PRx) may serve as marker of cerebral pressure autoregulation (CPA) and optimal CPP (CPPopt) may be assessed by identifying the CPP level with best (lowest) PRx. To evaluate the potential of CPPopt guided management in children with severe TBI, cerebral microdialysis (CMD) monitoring levels of lactate and the lactate/pyruvate ratio (LPR) (indicators of ischemia) were related to actual CPP levels, autoregulatory state (PRx) and deviations from CPPopt (ΔCPPopt). METHODS: Retrospective study of 21 children ≤ 17 years with severe TBI who had both ICP and CMD monitoring were included. CPP, PRx, CPPopt and ΔCPPopt where calculated, dichotomized and compared with CMD lactate and lactate-pyruvate ratio. RESULTS: Median age was 16 years (range 8-17) and median Glasgow coma scale motor score 5 (range 2-5). Both lactate (p = 0.010) and LPR (p = < 0.001) were higher when CPP ≥ 70 mmHg than when CPP < 70. When PRx ≥ 0.1 both lactate and LPR were higher than when PRx < 0.1 (p = < 0.001). LPR was lower (p = 0.012) when CPPopt ≥ 70 mmHg than when CPPopt < 70, but there were no differences in lactate levels. When ΔCPPopt > 10 both lactate (p = 0.026) and LPR (p = 0.002) were higher than when ΔCPPopt < -10. CONCLUSIONS: Increased levels of CMD lactate and LPR in children with severe TBI appears to be related to disturbed CPA (PRx). Increased lactate and LPR also seems to be associated with actual CPP levels ≥ 70 mmHg. However, higher lactate and LPR values were also seen when actual CPP was above CPPopt. Higher CPP appears harmful when CPP is above the upper limit of pressure autoregulation. The findings indicate that CPPopt guided CPP management may have potential in pediatric TBI.

DIEP flap perfusion assessment using microdialysis versus Doppler ultrasonography: A comparative study.

BACKGROUND: The increasing number of buried free-tissue transfer procedures and the need for an objective method to evaluate vascular complications of free flaps has led to the development of new technologies. Microdialysis has been used to monitor free flaps using interstitial biological markers. Previous uses mainly focused on muscular flaps. Our aim is to compare external Doppler ultrasonography (EDU) evaluation versus microdialysis in the early follow-up of adipocutaneous flaps, and propose an efficient postoperative monitoring protocol. METHODS: We retrospectively assessed 68 consecutive DIEP flaps (50 patients) performed between January 2019 and March 2021. All flaps received standardized post-operative monitoring using clinical signs, EDU and microdialysis. Glucose and lactate concentrations were assessed using glucose <1 mmol/L and lactate >6 mmol/L as ischemic trend thresholds. We calculated Glucose/Lactate ratio as a new parameter for the assessment of flap viability. RESULTS: Among all the 68 flaps, two flaps returned to the operative theater when a combination of unsatisfactory microdialysis values and clinical/EDU signs identified vascular impairment; only one developed total flap necrosis. Reoperation rate was 2.94% with an overall flap success rate of 98.53%. External Doppler ultrasonography had 100% sensitivity and 82% specificity, while microdialysis had 100% sensitivity and 100% specificity. CONCLUSIONS: Microdialysis values proved flap viability sooner than external Doppler ultrasonography, making it an excellent tool for post-operative monitoring. With the appropriate thresholds for glucose and lactate concentrations, and glucose/lactate ratio used as a new parameter, it can help potentially avoiding unnecessary re-explorations, and reducing flap ischemia times.

Cutaneous Pharmacokinetics of Topically Applied Novel Dermatological Formulations.

Novel formulations are developed for dermatological applications to address a wide range of patient needs and therapeutic challenges. By pushing the limits of pharmaceutical technology, these formulations strive to provide safer, more effective, and patient-friendly solutions for dermatological concerns, ultimately improving the overall quality of dermatological care. The article explores the different types of novel dermatological formulations, including nanocarriers, transdermal patches, microsponges, and microneedles, and the techniques involved in the cutaneous pharmacokinetics of these innovative formulations. Furthermore, the significance of knowing cutaneous pharmacokinetics and the difficulties faced during pharmacokinetic assessment have been emphasized. The article examines all the methods employed for the pharmacokinetic evaluation of novel dermatological formulations. In addition to a concise overview of earlier techniques, discussions on novel methodologies, including tape stripping, in vitro permeation testing, cutaneous microdialysis, confocal Raman microscopy, and matrix-assisted laser desorption/ionization mass spectrometry have been conducted. Emerging technologies like the use of microfluidic devices for skin absorption studies and computational models for predicting drug pharmacokinetics have also been discussed. This article serves as a valuable resource for researchers, scientists, and pharmaceutical professionals determined to enhance the development and understanding of novel dermatological drug products and the complex dynamics of cutaneous pharmacokinetics.

How can I measure brain acetylcholine levels in vivo? Advantages and caveats of commonly used approaches.

The neurotransmitter acetylcholine (ACh) plays a central role in the regulation of multiple cognitive and behavioral processes, including attention, learning, memory, motivation, anxiety, mood, appetite, and reward. As a result, understanding ACh dynamics in the brain is essential for elucidating the neural mechanisms underlying these processes. In vivo measurements of ACh in the brain have been challenging because of the low concentrations and rapid turnover of this neurotransmitter. Here, we review a number of techniques that have been developed to measure ACh levels in the brain in vivo. We follow this with a deeper focus on use of genetically encoded fluorescent sensors coupled with fiber photometry, an accessible technique that can be used to monitor neurotransmitter release with high temporal resolution and specificity. We conclude with a discussion of methods for analyzing fiber photometry data and their respective advantages and disadvantages. The development of genetically encoded fluorescent ACh sensors is revolutionizing the field of cholinergic signaling, allowing temporally precise measurement of ACh release in awake, behaving animals. Use of these sensors has already begun to contribute to a mechanistic understanding of cholinergic modulation of complex behaviors.

Comparison of ultrafiltration and microdialysis for ceftriaxone protein-binding determination.

BACKGROUND: High protein binding (PB) of antibiotics has an impact on their antimicrobial activity. It has been questioned whether in vitro PB determination can capture the dynamic and concentration-dependent PB of highly bound antibiotics. OBJECTIVES: This clinical study compared in vitro ultrafiltration (UF) and in vivo IV microdialysis (MD) methods to determine ceftriaxone PB. METHODS: Six healthy male volunteers received a single IV 2 g dose of ceftriaxone. Unbound ceftriaxone plasma concentrations were measured with MD and venous plasma sampling with subsequent UF. Pharmacokinetic parameters were determined using non-compartmental pharmacokinetic analysis. Non-linear mixed-effects modelling was used to quantify the PB. The PTA was estimated. RESULTS: The Cmax of ceftriaxone total plasma concentration (297.42 ±â€Š21.0 mg/L) was approximately 5.5-fold higher than for free concentrations obtained with UF (52.83 ±â€Š5.07 mg/L), and only 3.5-fold higher than for free concentrations obtained with MD (81.37 ±â€Š26.93 mg/L). Non-linear, saturable PB binding was confirmed for both UF and MD. Significantly different dissociation constants (Kd) for the albumin/ceftriaxone complex were quantified: in UF it was 23.7 mg/L (95% CI 21.3-26.2) versus 15.9 mg/L (95% CI 13.6-18.6) in MD. Moreover, the estimated number of binding sites (95% CI) per albumin molecule was 0.916 (0.86-0.97) in UF versus 0.548 in MD (0.51-0.59). The PTA obtained with MD was at most 27% higher than with UF. CONCLUSIONS: In vitro UF versus in vivo intravasal MD revealed significantly different PB, especially during the distribution phase. The method of PB determination could have an impact on the breakpoint determination and dose optimisation of antibiotics.

Involvement of nitric oxide synthase and reactive oxygen species in TRPA1-mediated cutaneous vasodilation in young and older adults.

OBJECTIVE: To investigate the nitric oxide synthase (NOS) and reactive oxygen species (ROS) contributions of the cutaneous vasodilator response to transient receptor potential ankyrin-1 channel (TRPA1) activation in young and older adults. MATERIALS AND METHODS: In sixteen young (20 ± 2 years, 8 females) and sixteen older adults (61 ± 5 years, 8 females), cutaneous vascular conductance normalized to maximum vasodilation (%CVCmax) was assessed at four dorsal forearm skin sites continuously perfused via microdialysis with: 1) vehicle solution (Control, 2 % dimethyl sulfoxide, 2 % Ringer, 96 % propylene glycol), 2) 10 mM Ascorbate (non-specific ROS inhibitor), 3) 10 mM L-NAME (non-specific NOS inhibitor), or 4) Ascorbate+L-NAME. The TRPA1 agonist cinnamaldehyde was co-administered at all sites [0 % (baseline), 2.9 %, 8.8 %, 26.4 %; ≥ 30 min per dose]. RESULTS: %CVCmax was not different between groups for Control, L-NAME, and Ascorbate (all p > 0.05). However, there were significant main dose effects for each site wherein %CVCmax was greater than baseline from 2.9 % to 26.4 % cinnamaldehyde for Control and Ascorbate, and at 26.4 % cinnamaldehyde for L-NAME and Ascorbate+L-NAME (all p < 0.05). For Ascorbate+L-NAME, there was a significant main group effect, wherein perfusion was 6 %CVCmax [95% CI: 2, 11, p < 0.05] greater in the older compared to the young group across all cinnamaldehyde doses. There was a significant main site effect for area under the curve wherein L-NAME and Ascorbate+L-NAME were lower than Control and Ascorbate across groups (all p < 0.05). CONCLUSION: The NOS-dependent cutaneous vasodilator response to TRPA1 activation is maintained in older adults, with no detectable contribution of ascorbate-sensitive ROS in either age group.

The Atypical Dopamine Transporter Inhibitor CE-158 Enhances Dopamine Neurotransmission in the Prefrontal Cortex of Male Rats: A Behavioral, Electrophysiological, and Microdialysis Study.

BACKGROUND: Dopamine plays a key role in several physiological functions such as motor control, learning and memory, and motivation and reward. The atypical dopamine transporter inhibitor S,S stereoisomer of 5-(((S)-((S)-(3-bromophenyl)(phenyl)methyl)sulfinyl)methyl)thiazole (CE-158) has been recently reported to promote behavioral flexibility and restore learning and memory in aged rats. METHODS: Adult male rats were i.p. administered for 1 or 10 days with CE-158 at the dose of 1 or 10 mg/kg and tested for extracellular dopamine in the medial prefrontal cortex by means of intracerebral microdialysis and single unit cell recording in the same brain area. Moreover, the effects of acute and chronic CE-158 on exploratory behavior, locomotor activity, prepulse inhibition, working memory, and behavioral flexibility were also investigated. RESULTS: CE-158 dose-dependently potentiated dopamine neurotransmission in the medial prefrontal cortex as assessed by intracerebral microdialysis. Moreover, repeated exposure to CE-158 at 1 mg/kg was sufficient to increase the number of active pyramidal neurons and their firing frequency in the same brain area. In addition, CE-158 at the dose of 10 mg/kg stimulates exploratory behavior to the same extent after acute or chronic treatment. Noteworthy, the chronic treatment at both doses did not induce any behavioral alterations suggestive of abuse potential (e.g., motor behavioral sensitization) or pro-psychotic-like effects such as disruption of sensorimotor gating or impairments in working memory and behavioral flexibility as measured by prepulse inhibition and Y maze. CONCLUSIONS: Altogether, these findings confirm CE-158 as a promising pro-cognitive agent and contribute to assessing its preclinical safety profile in a chronic administration regimen for further translational testing.

Active Uptake of Oxycodone at Both the Blood-Cerebrospinal Fluid Barrier and The Blood-Brain Barrier without Sex Differences: A Rat Microdialysis Study.

BACKGROUND: Oxycodone active uptake across the blood-brain barrier (BBB) is associated with the putative proton-coupled organic cation (H+/OC) antiporter system. Yet, the activity of this system at the blood-cerebrospinal fluid barrier (BCSFB) is not fully understood. Additionally, sex differences in systemic pharmacokinetics and pharmacodynamics of oxycodone has been reported, but whether the previous observations involve sex differences in the function of the H+/OC antiporter system remain unknown. The objective of this study was, therefore, to investigate the extent of oxycodone transport across the BBB and the BCSFB in female and male Sprague-Dawley rats using microdialysis. METHODS: Microdialysis probes were implanted in the blood and two of the following brain locations: striatum and lateral ventricle or cisterna magna. Oxycodone was administered as an intravenous infusion, and dialysate, blood and brain were collected. Unbound partition coefficients (Kp,uu) were calculated to understand the extent of oxycodone transport across the blood-brain barriers. Non-compartmental analysis was conducted using Phoenix 64 WinNonlin. GraphPad Prism version 9.0.0 was used to perform t-tests, one-way and two-way analysis of variance followed by Tukey's or Sídák's multiple comparison tests. Differences were considered significant at p < 0.05. RESULTS: The extent of transport at the BBB measured in striatum was 4.44 ± 1.02 (Kp,uu,STR), in the lateral ventricle 3.41 ± 0.74 (Kp,uu,LV) and in cisterna magna 2.68 ± 1.01 (Kp,uu,CM). These Kp,uu values indicate that the extent of oxycodone transport is significantly lower at the BCSFB compared with that at the BBB, but still confirm the presence of active uptake at both blood-brain interfaces. No significant sex differences were observed in neither the extent of oxycodone delivery to the brain, nor in the systemic pharmacokinetics of oxycodone. CONCLUSIONS: The findings clearly show that active uptake is present at both the BCSFB and the BBB. Despite some underestimation of the extent of oxycodone delivery to the brain, CSF may be an acceptable surrogate of brain ISF for oxycodone, and potentially also other drugs actively transported into the brain via the H+/OC antiporter system.

Reproducible Quantification of Unbound Fractions of Four Beta-Lactam Antibiotics: Ultrafiltration Versus Microdialysis of Spiked Healthy Donor Plasma.

BACKGROUND: Ultrafiltration (UF) is a conventional method for isolating the protein-unbound plasma fractions of therapeutic drugs. However, the ideal UF conditions for specific compounds remain largely unexplored. By comparing UF-derived unbound concentrations with the corresponding results obtained using a reference method, the authors sought to identify appropriate UF conditions for cefotaxime, cloxacillin, flucloxacillin, and piperacillin. METHODS: In vitro microdialysis (MD) with a no-net-flux approach was used as a reference method for plasma protein separation, for which UF performance was assessed. Four levels of relative centrifugal force (2500-11,290 g ) and 2 levels of temperature (37 vs. 22°C) during 10 minutes of UF centrifugation were evaluated. Ultrafiltrates and reference microdialysates were analyzed using liquid chromatography-tandem mass spectrometry to obtain unbound concentrations. After identifying the appropriate UF conditions in the spiked plasma samples, exploratory analyses of clinical samples (n = 10 per analyte) were performed. RESULTS: Of the evaluated UF alternatives, the best overall agreement with the MD-derived reference concentrations was obtained with 11,290 g UF performed at 22°C. For cloxacillin specifically, 37°C UF yielded better agreement than 22°C UF at 11,290 g. Clinical sample analyses indicated minimal differences between 22°C and 37°C at 11,290 g UF for cefotaxime and piperacillin. However, consistently lower levels of unbound cloxacillin (median: -23%, IQR: -19% to -24%) and flucloxacillin (median: -27%, IQR: -21 to -34%) were observed after UF at 22°C versus 37°C. CONCLUSIONS: For the evaluated UF device, 10 minutes of 11,290 g UF at 22°C is appropriate for flucloxacillin, cefotaxime, and piperacillin, and can arguably be justified for cloxacillin as well for laboratory practice purposes. Maintenance of 37°C during high-centrifugal UF may lead to overestimation, particularly for unbound flucloxacillin.

Application of microdialysis combined with UHPLC-QTOF/MS to screen for endogenous metabolites in aquatic organisms as biomarkers of exposure to an emerging contaminant, triclosan.

The detection of emerging contaminants (ECs) and understanding their ecotoxicity has brought new challenges to water pollution control. Triclosan (TCS), as an emerging contaminant, is a commonly used antibacterial agent widely present in the environment. Microdialysis (MD), as a sampling technique, can overcome some of the deficiencies of traditional approaches to sampling, using sources such as blood, urine, tissue, and target organs, in terms of invasiveness, time from collection to analysis, and possible changes during sample preparation. In this study, we coupled MD with analysis using UHPLC-QTOF/MS to identify the endogenous metabolites in the liver as biomarkers of the exposure of living crucian carp to TCS. The identified biomarkers were then quantified using UHPLC-MS/MS to continuously monitor the effect of TCS on endogenous metabolites in the liver of living crucian carp, which contributes to a better understanding of the toxicological effect of TCS. The experimental results demonstrated that TCS exposure interfered with the metabolic pathways of amino acids (L-isoleucine and L-histidine), purines (xanthine and hypoxanthine), and small nerve molecules (acetylcholine and choline).

Comparative pharmacokinetics of four bioactive components in normal and chronic heart failure rats after oral administration of Qiangxin Lishui Prescription by microdialysis combined with ultra-high-performance liquid chromatography.

Qiangxin Lishui Prescription (QLP) has been clinically applied for treating heart failure with remarkable curative effects. A multi-component pharmacokinetic research is very necessary for determining active substances in it. This study aims to profile the traits and differences in the pharmacokinetics of salvianolic acid B, astragaloside IV, calycosin-7-O-ß-D-glucoside and kaempferol in QLP between normal and chronic heart failure (CHF) rats by microdialysis combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Sensitive, selective, and online microdialysis combined with the UHPLC-MS/MS method was successfully established and applied to study the pharmacokinetics of QLP. The pathological condition of CHF could lead to the enhancement of systematic exposure and reduction of the metabolic rate of four bioactive components for better bioavailability and therapeutic efficacy. The pharmacokinetic results will provide data support for the clinical application of QLP.

Regulation of ethanol-mediated dopamine elevation by glycine receptors located on cholinergic interneurons in the nucleus accumbens.

Alcohol use disorder is one of the major psychiatric disorders worldwide, and there are many factors and effects contributing to the disorder, for example, the experience of ethanol reward. The rewarding and reinforcing properties of ethanol have been linked to activation of the mesolimbic dopamine system, an effect that appears to involve glycine receptors (GlyRs) in the nucleus accumbens. On which neuronal subtypes these receptors are located is, however, not known. The aim of this study was to explore the role of GlyRs on cholinergic interneurons (CIN) in sustaining extracellular dopamine levels and in ethanol-induced dopamine release. To this end, CIN were ablated by anti-choline acetyltransferase-saporin administered locally in the nucleus accumbens of male Wistar rats. Changes in dopamine levels induced by ablation, ethanol and/or a GlyR antagonist were monitored using in vivo microdialysis. The GlyRs antagonist strychnine depressed extracellular dopamine in a similar manner independent on local ablation, suggesting that GlyRs on CIN are not important for sustaining the extracellular dopamine tone. However, a low concentration of strychnine hampered ethanol-induced dopamine release in sham-treated animals, whilst no reduction was seen in ablated animals, suggesting that GlyRs located on CIN are involved in ethanol-induced dopamine release. Further, in ablated rats, ethanol-induced increases of the extracellular levels of the GlyR agonists glycine and taurine were attenuated. In conclusion, this study suggests that CIN are not important for GlyR-mediated regulation of basal dopamine output, but that CIN ablation blunts the ethanol-induced dopamine release, putatively by reducing the release of GlyR agonists.