Free interstitial levels of metformin in the liver of healthy and diabetic Wistar rats

Abstract In the present study, free interstitial levels reached by metformin in the liver were investigated in control and diabetic rats by microdialysis. Firstly, a bioanalytical method using an HPLC-UV system to determine the drug concentration in microdialysis samples was validated. The blood glucose levels and biochemical parameters were investigated in control and diabetic animals. Following that, both groups received a dose of 50 mg/kg of metformin iv bolus and the free interstitial levels reached in the liver were assessed by microdialysis. The method was validated according to FDA guidelines being suitable to quantify free concentrations of metformin in the liver of control and diabetics rats. Free exposure to metformin was similar in control and diabetic animals: AUC0-∞ 118.50 ± 40.18 vs 112.93 ± 50.25 µg.h/mL, respectively. The half-life in tissue was similar to that described in the literature for plasma. Hence diabetes induced by streptozotocin after administration of nicotinamide in our study did not damage the renal and hepatic function of the animals. The levels reached in the liver were 1.6 times higher than the free plasma concentrations, demonstrating higher liver penetration of metformin. This is the first investigation in liver interstitial concentration of metformin in control and diabetic rats

Metabonomics research strategy based on microdialysis technique / 中国中药杂志

Metabonomics is the branch of systems biology. It has been widely used in the fields of diagnostic markers discovery, disease prognosis, drug action mechanism, drug efficacy and toxicity evaluation, traditional Chinese medicine syndromes differentiation. There are shortcomings in the conventional metabonomics research. Microdialysis technology is a new type of biosampling technology, and metabonomics research based on microdialysis technology is in the ascendant. In view of the particularity of microdialysis technology and its great differences from traditional sampling and pretreatment methods, the metabonomics process based on microdialysis technology has certain similarities with traditional metabonomics research, and its basic process has some particularity. Advantages and basic strategies of metabonomics research by microdialysis technology are systematically summarized for researchers' reference.

Combination of In Vivo ¹²³IFP-CIT SPECT and Microdialysis Reveals an Antipsychotic Drug Haloperidol-induced Synaptic Dopamine Availability in the Rat Midbrain and Striatum

Synaptic dopamine (DA) is mainly regulated by the presynaptic DA transporter (DAT). Single-photon emission computerized tomography (SPECT) with the DAT radiotracer [¹²³I]FP-CIT assesses changes in synaptic DA availability when endogenous DA displaces [¹²³I]FP-CIT or competes for DAT. Here, we investigated the effects of haloperidol (HAL) and clozapine (CLZ) on [¹²³I]FP-CIT binding in the rat striatum and midbrain to assess the utility of [¹²³I]FP-CIT SPECT to quantify changes in synaptic DA availability. Rats underwent [¹²³I]FP-CIT SPECT after intraperitoneal administration of normal saline (vehicle), HAL (1 and 7 mg/kg), CLZ (10 and 54 mg/kg) and bupropion (BUP, a DAT blocker, 20 and 100 mg/kg). In the striatum and midbrain, percent differences in the nondisplaceable binding potential (BP(ND)) of [¹²³I]FP-CIT compared to the vehicle were calculated for the various drugs and doses. In another experiment, changes in endogenous striatal DA concentration were measured by in vivo microdialysis under the conditions used in the SPECT study. BUP dose-dependently occupied DAT at considerable levels. Compared to the vehicle, HAL decreased [¹²³I]FP-CIT BP(ND) in the striatum (−25.29% and −2.27% for 1 and 7 mg/kg, respectively) and to a greater degree in the midbrain (−58.74% and −49.64% for 1 and 7 mg/kg, respectively), whereas the CLZ-treated group showed a decrease in the midbrain (−38.60% and −40.38% for 10 and 54 mg/kg, respectively) but an increase in the striatum (18.85% and 38.64% for 10 and 54 mg/kg, respectively). Antipsychotic-induced changes in endogenous striatal DA concentrations varied across drugs and doses. The data demonstrate that [¹²³I]FP-CIT SPECT may be a useful preclinical technique for detecting increases in synaptic DA availability in the midbrain and striatum in response to HAL, with results comparable to those of in vivo microdialysis.

Change of Extracellular Glutamate Level in Striatum during Deep Brain Stimulation of the Entopeduncular Nucleus in Rats

OBJECTIVE: Globus pallidus interna (GPi) is acknowledged as an essential treatment for advanced Parkinson’s disease (PD). Nonetheless, the neurotransmitter study about its results is undiscovered. The goal of this research was to examine influences of entopeduncular nucleus (EPN) stimulation, identical to human GPi, in no-lesioned (NL) rat and 6-hydroxydopamine (6-HD)-lesioned rat on glutamate change in the striatum. METHODS: Extracellular glutamate level changes in striatum of NL category, NL with deep brain stimulation (DBS) category, 6-HD category, and 6-HD with DBS category were examined using microdialysis and high-pressure liquid chromatography. Tyrosine hydroxylase (TH) immunoreactivities in substantia nigra and striatum of the four categories were also analyzed. RESULTS: Extracellular glutamate levels in the striatum of NL with DBS category and 6-HD with DBS category were significantly increased by EPN stimulation compared to those in the NL category and 6-HD category. EPN stimulation had no significant effect on the expression of TH in NL or 6-HD category. CONCLUSION: Clinical results of GPi DBS are not only limited to direct inhibitory outflow to thalamus. They also include extensive alteration within basal ganglia.

Change of Extracellular Glutamate Level in Striatum during Deep Brain Stimulation of the Entopeduncular Nucleus in Rats

OBJECTIVE: Globus pallidus interna (GPi) is acknowledged as an essential treatment for advanced Parkinson’s disease (PD). Nonetheless, the neurotransmitter study about its results is undiscovered. The goal of this research was to examine influences of entopeduncular nucleus (EPN) stimulation, identical to human GPi, in no-lesioned (NL) rat and 6-hydroxydopamine (6-HD)-lesioned rat on glutamate change in the striatum.METHODS: Extracellular glutamate level changes in striatum of NL category, NL with deep brain stimulation (DBS) category, 6-HD category, and 6-HD with DBS category were examined using microdialysis and high-pressure liquid chromatography. Tyrosine hydroxylase (TH) immunoreactivities in substantia nigra and striatum of the four categories were also analyzed.RESULTS: Extracellular glutamate levels in the striatum of NL with DBS category and 6-HD with DBS category were significantly increased by EPN stimulation compared to those in the NL category and 6-HD category. EPN stimulation had no significant effect on the expression of TH in NL or 6-HD category.CONCLUSION: Clinical results of GPi DBS are not only limited to direct inhibitory outflow to thalamus. They also include extensive alteration within basal ganglia.

Pharmacokinetics of Achyranthes bidentata on adjuvant arthritis rats by microdialysis and UHPLC-MS/MS / 中国中药杂志

To investigate the " drug-guide" effect of Achyranthes bidentata saponins( ABS) and geniposide( GE) in the treatment on adjuvant arthritis( AA) rats. A UHPLC-MS/MS method for the quantitative determination of GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa in rat blood and joint dialysate was established. After single or combined administration with ABS and GE was given to AA rat model,a microdialysis sampling method for rat joint cavity and jugular vein blood vessels was established to collect microdialysis samples. Waters Acquity HSS C_(18) column was used to separate the above four components,with mobile phase as acetonitrile-0. 1% formic acid water as mobile phase for gradient elution. ESI source was adopted for mass spectra in a negative ion scanning mode. Multiple reaction monitoring( MRM) mode was applied to detect the above four components. The methodological results showed that GE,zingibroside R1,ginsenoside Ro and chikusetsu saponin Ⅳa demonstrated a good linear relationship within the concentration ranges of 2-4 000,16-4 096,14-3 584,23-5 888 μg·L-1 respectively. The precision,accuracy,stability and matrix effect of these four ingredients reached the requirements of quantitative analysis of biological samples. The pharmacokinetic results demonstrated that the combined administration of ABS and GE( 60 mg·kg~(-1)+60 mg·kg~(-1)) can increase the degree of GE in joint cavity distribution,and the AUCjoint/AUCplasmwere twice of that of single administration of GE( 60 mg·kg~(-1)),which indicated that ABS might played a vital role in GE's distribution to joint cavity. Moreover,there was no significant difference between the distribution trend of total three ABS and GE in rats. The pharmacodynamics results showed that the combined administration of ABS and GE has stronger effects on paw swelling,arthritis index and synovial pathomorphology of AA rats than single administration of GE,which suggested that ABS might improve GE's anti-inflammatory effect in AA rats. Based on the above results,ABS has a targeting effect in increasing GE's concentration in joint cavity,with a synergy in efficacy.

Experimental factors affecting recovery of puerarin in microdialysis / 浙江大学学报·医学版

OBJECTIVE@#: To analyze experimental factors affecting recovery of puerarin in microdialysis.@*METHODS@#: Puerarin concentration in microdialysate samples was determined by high performance liquid chromatography. The methods of direct dialysis, retrodialysis and the zero-net flux were used to calculate recovery, respectively. The effects of perfusate composition, the analyte concentration, perfusate flow rate, medium temperature and stir rates of the dialysis medium on recovery were investigated.@*RESULTS@#: There were significant differences in the recovery values among direct dialysis, retrodialysis and zero-net flux methods. The recovery for 0.9% NaCl solution, Ringer's solution, PBS and anticoagulant dextrose solution as perfusate fluid were (71.25±2.36)%,(73.48±1.41)%,(68.50±2.43)% and (74.98±1.16)%, respectively. The composition of perfusate fluid had significant influence on the recovery(<0.01). At the same flow rate, recovery was independent of the analyte concentration. At the same concentration, the recovery was decrease with the increasing flow rate in an exponential relationship. The recovery increased with the raising temperature and stir rate of the dialysis medium, and the recovery remained stable when the stir rate reached above 200 rpm.@*CONCLUSIONS@#: A study method for recovery of puerarin in microdialysis has been established, and the recovery of puerarin is affected by calculating methods, perfusate fluids, flow rate, medium temperature and stir rate, but not affected by analyte concentrations.

Striatal Glutamate and GABA after High Frequency Subthalamic Stimulation in Parkinsonian Rat

OBJECTIVE: High frequency stimulation (HFS) of the subthalamic nucleus (STN) is recognized as an effective treatment of advanced Parkinson’s disease. However, the neurochemical basis of its effects remains unknown. The aim of this study is to investigate the effects of STN HFS in intact and 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian rat model on changes of principal neurotransmitters, glutamate, and gamma-aminobutyric acid (GABA) in the striatum. METHODS: The authors examined extracellular glutamate and GABA change in the striatum on sham group, 6-OHDA group, and 6-OHDA plus deep brain stimulation (DBS) group using microdialysis methods. RESULTS: High-pressure liquid chromatography was used to quantify glutamate and GABA. The results show that HFS-STN induces a significant increase of extracellular glutamate and GABA in the striatum of 6-OHDA plus DBS group compared with sham and 6-OHDA group. CONCLUSION: Therefore, the clinical results of STN-HFS are not restricted to the direct STN targets but involve widespread adaptive changes within the basal ganglia.

Extracellular adenosine 5'-triphosphate concentrations changes in rat spinal cord associated with the activation of urinary bladder afferents. A microdialysis study. / Variação da concentração extracelular de 5'-trifosfato de adenosina na medula espinal de rata associadas a fibras aferentes vesicais. Um estudo com microdiálise

ABSTRACT Objective To determine adenosine 5’-triphosphate levels in the interstice of spinal cord L6-S1 segment, under basal conditions or during mechanical and chemical activation of urinary bladder afferents. Methods A microdialysis probe was transversally implanted in the dorsal half of spinal cord L6-S1 segment in female rats. Microdialysate was collected at 15 minutes intervals during 135 minutes, in anesthetized animals. Adenosine 5’-triphosphate concentrations were determined with a bioluminescent assay. In one group of animals (n=7) microdialysate samples were obtained with an empty bladder during a 10-minutes bladder distension to 20 or 40cmH2O with either saline, saline with acetic acid or saline with capsaicin. In another group of animals (n=6) bladder distention was performed and the microdialysis solution contained the ectonucleotidase inhibitor ARL 67156. Results Basal extracellular adenosine triphosphate levels were 110.9±35.34fmol/15 minutes, (mean±SEM, n=13), and bladder distention was associated with a significant increase in adenosine 5’-triphosphate levels which was not observed after bladder distention with saline solution containing capsaicin (10µM). Microdialysis with solution containing ARL 67156 (1mM) was associated with significantly higher extracellular adenosine 5’-triphosphate levels and no further increase in adenosine 5’-triphosphate was observed during bladder distension. Conclusion Adenosine 5’-triphosphate was present in the interstice of L6-S1 spinal cord segments, was degraded by ectonucleotidase, and its concentration increased following the activation of bladder mechanosensitive but not of the chemosensitive afferents fibers. Adenosine 5’-triphosphate may originate either from the central endings of bladder mechanosensitive primary afferent neurons, or most likely from intrinsic spinal neurons, or glial cells and its release appears to be modulated by capsaicin activated bladder primary afferent or by adenosine 5’-triphosphate itself.

RESUMO Objetivo Determinar as concentrações extracelulares do 5’-trifosfato de adenosina no interstício dos segmentos medulares L6-S1, em condições basais ou durante a ativação mecânica e química das fibras aferentes vesicais. Métodos Um cateter de microdiálise foi implantado no sentido transversal na parte dorsal da medula espinal, entre os segmentos L6-S1 de ratas. O microdialisado foi coletado em intervalos de 15 minutos, durante 135 minutos, com os animais anestesiados. A concentração de 5’-trifosfato de adenosina nas amostras foi determinada mediante ensaio de bioluminescência. Em um grupo de animais (n=7), as amostras de microdialisado foram obtidas com a bexiga vazia, com distensão da bexiga para volume de 20 ou 40cmH2O, com solução salina, solução salina com ácido acético, ou solução salina com capsaicina. Em outro grupo (n=6), foi realizada com a bexiga distendida, e a solução para microdiálise continha o inibidor de ectonucleotidase ARL 67156. Resultados Os níveis extracelulares de trifosfato de adenosina no início do estudo foram 110,9±35,36fmol/15 minutos (média±EPM, n=13), e a distensão da bexiga causou um aumento nos níveis de 5’-trifosfato de adenosina, o que não foi observado após a distensão da bexiga com solução salina contendo capsaicina (10µM). A microdiálise com solução contendo ARL 67156 (1mM) foi associada com significante aumento dos níveis de trifosfato de adenosina extracelular, e nenhum aumento do trifosfato de adenosina foi observado durante a distensão da bexiga. Conclusão O 5’-trifosfato de adenosina está presente no interstício do segmento L6-S1 da medula espinal, é degradado por ectonucleotidases, e sua concentração aumentou com a ativação das fibras aferentes mecanossensíveis da bexiga, mas não das quimiossensíveis. O 5’-trifosfato de adenosina pode ter sido liberado das terminações centrais dos neurônios aferentes primários mecanossensíveis ou, mais provavelmente, de neurônios espinais intrínsecos, ou ainda de células gliais. Sua liberação parece ser modulada por fibras aferentes primárias da bexiga ativadas pela capsaicina ou pelo próprio 5’-trifosfato de adenosina.

The Role of Spinal Dopaminergic Transmission in the Analgesic Effect of Nefopam on Rat Inflammatory Pain

BACKGROUND: Nefopam has been known as an inhibitor of the reuptake of monoamines, and the noradrenergic and/or serotonergic system has been focused on as a mechanism of its analgesic action. Here we investigated the role of the spinal dopaminergic neurotransmission in the antinociceptive effect of nefopam administered intravenously or intrathecally. METHODS: The effects of intravenously and intrathecally administered nefopam were examined using the rat formalin test. Then we performed a microdialysis study to confirm the change of extracellular dopamine concentration in the spinal dorsal horn by nefopam. To determine whether the changes of dopamine level are associated with the nefopam analgesia, its mechanism was investigated pharmacologically via pretreatment with sulpiride, a dopaminergic D2 receptor antagonist. RESULTS: When nefopam was administered intravenously the flinching responses in phase I of the formalin test were decreased, but not those in phase II of the formalin test were decreased. Intrathecally injected nefopam reduced the flinching responses in both phases of the formalin test in a dose dependent manner. Microdialysis study revealed a significant increase of the level of dopamine in the spinal cord by intrathecally administered nefopam (about 3.8 fold the baseline value) but not by that administered intravenously. The analgesic effects of intrathecally injected nefopam were not affected by pretreatment with sulpiride, and neither were those of the intravenous nefopam. CONCLUSIONS: Both the intravenously and intrathecally administered nefopam effectively relieved inflammatory pain in rats. Nefopam may act as an inhibitor of dopamine reuptake when delivered into the spinal cord. However, the analgesic mechanism of nefopam may not involve the dopaminergic transmission at the spinal level.

Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe / 中国中药杂志

To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 μL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo.

In vitro microdialysis recoveries of nine active ingredients in Mahuang decoction / 中国中药杂志

To detect the in vitro probe microdialysis recoveries based on an HPLC-DAD method for simultaneous quantification of nine active ingredients (ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid) in Mahuang decoction, which provides reference for in vivo pharmacokinetic study. The concentrations of nine active ingredients in dialysate were detected by HPLC-DAD, to investigate the effect of flow rates (incremental method and subtraction method) and intraday stability of the probe recoveries and medium concentrations on the recoveries. Nine active ingredients could be well separated in 52 min. At the perfusion rate of 1.0 μL x min(-1), the relative recoveries of ephedrine, pseudoephedrine, methylephedrine, amygdalin, liquiritin, cinnamyl alcohol, cinnamic acid, cinnamaldehyde and glycyrrhizic acid were (50.95 ± 0.82)%, (52.74 ± 1.13)%, (51.29 ± 0.51)%, (32.56 ± 0.84)%, (45.36 ± 0.83)%, (70.94 ± 0.99)%, (69.98 ± 2.30)%, (71.68 ± 0.63)%, and (22.14 ± 0.48)%, respectively. And the probe kept steady in 7 hours. At the same medium concentration, the probe recoveries decreased exponentially with the increase in flow rates. The recoveries of seven ingredients detected by these two methods were similar at certain flow rates, except for amygdalin and cinnamaldehyde. At the same flow rate, the relative recoveries of cinnamyl alcohol, cinnamic acid and cinnamaldehyde changed greatly (9.55%-16.2%) and the others six ingredients had less change (3.27%-5.71%) with the changes in medium concentrations. Microdialysis method could be used to detect the in vitro recoveries of nine ingredients in Mahuang decoction. Reverse dialysis method could be used for the in vivo probe recovery calibration of ephedrine, pseudoephedrine, methylephedrine, liquiritin, cinnamyl alcohol and cinnamic acid at the flow rate of 2.0 μL x min(-1).

Research progress in the study of brain microdialysis in glioma / 药学学报

Glioma is the most common form of brain cancer. Despite recent advances in the treatment of solid tumors, there are few effective treatments for malignant gliomas due to its infiltrative nature. It has important significance to improve the treatment of glioma through in-depth understanding the intracerebral metabolic characteristics and pharmacokinetics of chemotherapeutics. Brain microdialysis (B-MD), an effective method to monitor central nervous system anticancer drug disposition, conditions of drugs through the blood-brain barrier, basic pathophysiologic metabolism, bioactive compounds and the changes of neurotransmitter in brain, provides the unique opportunity to allow the simultaneous determination of unbound concentrations of drugs in several tissues, and directly measure gliomas biochemistry continuously. B-MD has been able to monitor the change of brain drugs, metabolites and neurotransmitters, dynamic analysis of the drug concentration and pharmacological effect after administration, pharmacodynamic interaction between drugs, receptor mechanism of drug transport, as well as feedback information of internal environment. B-MD is expected to provide reference for clinical individual chemotherapy of glioma, but also provide powerful tools for the evaluation of new anticancer drugs in vivo. In this review, a comprehensive overview of B-MD for studies on glioma is elucidated with special emphasis on its application to neurochemistry and pharmacokinetic studies.

Dynamics of interstitial calcium in rat myocardial ischemia reperfusion injury in vivo / 华中科技大学学报（医学）（英德文版）

Intracellular calcium overload is a key factor for myocardial ischemia reperfusion injury (IR). However, there was no report for interstitial calcium concentration dynamics. We investigated the interstitial calcium dynamics in rat myocardial IR model in vivo. A microdialysis system was involved, and the time delay of the system and recovery time was introduced and tested with a fluids switching method. Twelve SD rats were divided into IR or control group. Myocardial IR was induced by ligating (20 min) then releasing (60 min) the suture underlying left anterior descending branch. Mycrodialyisis probe was implanted into the left ventricular myocardium perfusion area for occlusion. Dialysate samples were collected every 10 min. Dialysate calcium concentration was detected with an atomic absorption spectrophotometer. Recovery time for the microdialysis system was 20 min, and recovery rate was 16%. Dialysate calcium concentration showed no changes during ischemia, descended immediately after reperfusion, reached the lowest level (67% of baseline value) 20 min after reperfusion, then escalated slowly. Recovery time was an important parameter for mycrodialysis technique, and it should not be neglected and needed to be tested. Our data suggest that interstitial calcium concentration in rats with myocardial IR in vivo kept steady in ischemia, descended rapidly at the initial reperfusion, then rebounded slowly. In conclusion, we introduced the concept of recovery time for microdialysis and provided a simple testing method.

Preparation of paeonol transdermal delivery systems based on proniosomes-based ointment and its pharmacokinetics characters / 中国中药杂志

The paeonol proniosomes ointment and ordinary ointment were administered to rats. Physiological saline served as perfused solution. The perfusion rate was 5 mL x L(-1) and the microdialysis samples were collected every 20 min intervals. The paeonol concentration in perfused solution was determined by HPLC. Investigation of the pharmacokinetics of paeonol proniosomes ointment and ordinary ointment by the skin-blood synchronous microdialysis coupled with HPLC is reported in this study. The results show that the recovery was (54.80 +/- 1.50)% in vitro and (54.58 +/- 4.61)% in vivo. The results showed that paeonol proniosomes ointment significantly raised the drug concentrations in skin more than the paeonol ordinary ointment. The paeono proniosomes ointment has less drugs into the blood as the ordinary ointments in blood, but its blood drug concentrations were steadier. The paeonol proniosomes ointment may be developed into a new preparation.

Metabólitos de óxido nítrico no interstício da medula espinhal lombossacral e no líquido cefalorraquidiano em ratas com cistite aguda induzida por ciclofosfamida: um estudo in vivo com microdiálise / Nitric oxide metabolites in the lumbosacral spinal cord interstice and cerebrospinal fluid in female rats with acute cyclophosphamide-induced cystitis: an in vivo microdialysis study

OBJETIVO: Determinar a concentração de nitrato/nitrito no líquido cefalorraquidiano e no interstício do corno dorsal entre L6-S1 da medula espinhal em ratas com ou sem cistite induzida por ciclofosfamida. MÉTODOS: Todos os experimentos foram conduzidos usando ratas Wistar. Um probe de microdiálise foi implantado no espaço subaracnoide ou no tecido da medula espinhal nos segmentos L6-S1 (confirmado histologicamente). Dois dias depois, o probe de microdiálise foi perfundido com líquido cefalorraquidiano artificial, contendo ou não NG-monometil-L-arginina. As amostras foram coletadas a cada 15 minutos e mantidas a -20ºC. As concentrações de nitrito/ nitrato foram determinadas por quimiluminescência. RESULTADOS: Nos animais normais, os valores médios das concentrações de nitrito/nitrato, na primeira amostra de microdialisado de líquido cefalorraquidiano e do interstício da medula espinhal, foram semelhantes (482,5±90,2pmol/75µL, n=20, e 505,7±11,5pmol/75µL, n=6, respectivamente), enquanto nas amostras de ratas com cistite, esses valores foram significativamente maiores (955,5±66,3pmol/75µL, n=8, e 926,5±131,7pmol/75µL, n=11, respectivamente). Em ambos os grupos, a NG-monometil-L- arginina causou uma significativa redução na concentração de nitrito/nitrato. Curiosamente, a redução máxima de concentração de nitrito/nitrato causada pela NG-monometil-L- arginina não foi maior que 30% dos valores iniciais. CONCLUSÕES: Esses resultados constituem a primeira demonstração de que as concentrações de nitrito/nitrato no líquido cefalorraquidiano e no interstício da medula espinhal estão elevadas entre 20 e 22 horas após a cistite induzida por ciclofosfamida, e indicando que a cistite está associada a alterações na produção de óxido nítrico, nos segmentos da medula espinhal, nos quais termina a maioria dos aferentes primários da bexiga.

OBJECTIVE: To determine the concentration of nitrate/nitrite in the cerebrospinal fluid and in the dorsal horn interstice of the L6-S1 spinal cord boundary in rats with or without cystitis induced by cyclophosphamide. METHODS: All experiments were conducted using Wistar female rats. A microdialysis probe was implanted in the subarachnoid space or in the spinal cord tissue at the L6-S1 segments (confirmed histologically). Two days later, the microdialysis probe was perfused with artificial cerebrospinal fluid, containing or not NG-monomethyl-L-arginine. Samples were collected every 15 minutes and kept at -20ºC. Nitrite/nitrate concentrations were determined by chemiluminescence. RESULTS: In normal animals, the mean values of nitrite/nitrate concentrations in the first microdialysate sample of the cerebrospinal fluid and of the spinal cord interstice were similar (482.5±90.2pmol/75µL, n=20, and 505.7±11.5pmol/75µL, n=6, respectively), whereas, in the samples from rats with cystitis, these values were significantly greater (955.5±66.3pmol/75µL, n=8, and 926.5±131.7pmol/75µL, n=11, respectively). In both groups, NG-monomethyl-L- arginine caused a significant reduction in the nitrite/nitrate concentration. Interestingly, the maximal reduction of nitrite/nitrate concentration caused by NG-monomethyl-L- arginine was no greater than 30% of the initial values. CONCLUSIONS: These results constitute the first demonstration that nitrite/nitrate concentrations in the cerebrospinal fluid and spinal cord interstice are elevated between 20- and 22 hours after cyclophosphamide-induced cystitis, and indicate that cystitis is associated with changes in the production of nitric oxide in the spinal cord segments, where most primary bladder afferents end.

Significance of Bioelectrical Impedance Change after Ischemia and Reperfusion Injury in Liver and What it Causes? / 한양의대학술지

PURPOSE: Ischemia and reperfusion (I/R) injury is a major cause of hepatic failure after liver surgery, but there is no direct method to monitor it in real-time (like an ECG in heart disease) during surgery. Recently we found the possible role of bioelectrical impedance (BEI) to monitor I/R injury in liver, but the mechanism responsible for ischemia-related BEI changes has not been clearly determined. METHODS: The authors used a LCR meter to quantify BEI changes at 0.12 KHz. Livers were subjected to 70% partial ischemia for 120 minutes, and ATP content, cation changes in extracellular fluid (ECF; determined using an in vivo intracellular microdialysis technique), hepatocyte sizes, and histological changes were then examined. RESULTS: Liver tissue BEI was found to increase gradually during the first 60 minutes of ischemia and then tended to plateau. During the same period, intracellular ATP content decreased to below 20% of the baseline level, [Na+] in ECF decreased from 150.4+/-3.8 to 97.8+/-10.6 mmol/L, and [K+] in ECF increased from 7.5+/-0.3 to 34.3+/-5.5 mmol/L during the first 60 minutes of ischemia. Hepatocyte diameter increased by approximately 20% during the first 60 minutes of ischemia. CONCLUSION: This study suggests that BEI changes during hepatic ischemia are probably caused by sodium and potassium concentration changes in the ECF due to reduced intracellular ATP content.

Effects of ligustrazine on extracellular dopamine levels in rat brain dialysate / 药学学报

Using brain microdialysis and LC-ECD, the content of dopamine in rat brain was detected to investigate the effects of ligustrazine. A liquid chromatography-electrochemical detector method has been established and validated for the determination of dopamine in rat brain dialysate. The results indicate that ligustrazine administration by subcutaneous injection significantly increased dopamine release in rat medial prefrontal cortex, nucleus accumbens and hippocampus in a dose-related manner. The drug's effects on dopa release in rat brain could be directly detected by microdialysis combined with HPLC-ECD and this method has the preponderance over traditional neurology methods.

Determination of 7-ethyl-10-hydroxycamptothecin in microdialysates from rat brain with LC-MS/MS / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a method for determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in microdialysates from rat brain.</p><p><b>METHODS</b>The concentrations of SN-38 were measured by LC-MS/MS method with Agilent Eclipse Plus C18 (2.1 mm ×100 mm, 1.8 μm) reversed phase column using acetonitrile-0.1% methanoic acid as mobile phase with gradient elution at a flow rate of 0.3 ml/min and temperature at 35 degree. Multiple reaction monitoring using the precursor to product ion combinations of m/z 393.1→349.1 was performed to detect SN-38 in microdialysates from rat brain.</p><p><b>RESULTS</b>Blank microdialysate had non-interference. The method was linear over the concentration range of 0.1015-1015 ng/ml (r=0.9995); and the lower limit of quantification (LOQ) was 0.1015 ng/ml. The recovery of assay for SN-38 ranged from 97.54%-100.60%. The intra- and inter-day precision and stability were both well. The concentrations of SN-38 in brain microdialysates presented pharmacokinetics process and achieved the peak after 220 min.</p><p><b>CONCLUSION</b>The fully validated LC-MS/MS analytical method has high specificity and sensibility, which can be used effectively to analyze SN-38 in microdialysates from rat brain.</p>

Pharmacokinetics of tramadol hydrochloride in the extracellular fluid of mouse frontal cortex studied by in vivo microdialysis / 药学学报

The paper aims to explore the studying method for the pharmacokinetics of drugs in target organs, the pharmacokinetic process of tramadol hydrochloride in the extracellular fluid of frontal cortex (FrCx) of mice was investigated. Six male mice (Kunming strain) were anaesthetized (urethane, 1.8 g x kg(-1), ip) and secured on a stereotaxic frame. A microdialysis probe was implanted into the FrCx and perfused with artificial cerebrospinal fluid at a flow rate of 2 microL x min(-1). One hour later, mice were administrated (ip) with tramadol hydrochloride (50 mg x kg(-1)) and dialysates were collected continuously at 12-min intervals (24 microL each) for 6 h. The tramadol concentration in dialysates was determined by HPLC-Ultraviolet detection method, and the concentration-time curve and pharmacokinetic parameters of tramadol were calculated with DAS software. The results showed that the pharmacokinetic process of tramadol in the FrCx extracellular fluid of mice was fitted to a two-compartment open model, and the main pharmacokinetic parameters t1/2alpha, t1/2beta, t(max), C(max) and AUC(0-infinity) were (0.27 +/- 0.05) h, (2.72 +/- 0.24) h, (0.50 +/- 0.10) h, (2 110.37 +/- 291.22) microg x L(-1) and (4 474.51 +/- 441.79) microg x L(-1) x h, respectively. In conclusion, a studying method for pharmacokinetics of drugs in the target organ is established, which is simple and feasible. Tramadol hydrochloride shows a two-compartment model in the extracellular fluid of the mouse FrCx, and the distribution- and elimination half-life are 0.5 h and 2.7 h, respectively.