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In vivo applicability of brain microdialysis in neuropharmacological investigation
82
Pharmacological Research, Vol . 25, Supplement 2, 1992
IN VIVO APPLICABILITY OF BRAIN MICRODIALYSIS IN NEUROPHARMACOLOGICAL INVESTIGATION
J . Tarcali, K . Magyar Department of Pharmacodynamics Semmelweis University of Medicine, Budapest, Hungary Key
words :
in vivo, brain deprenyl, reserpine
microdialysis,
hplc-ec,
Introduction : One of the most promising progress in experimental neuropharmacology in recent years have been achieved in "in vivo" monitoring of extracellular concentrations of neuroactive compounds . Ungerstedt and Justice et al developed flow-trough micro samplers based on single dialysis fiber have been invented for in vivo sample collection (1, 2, 3) . These samplers can be implanted with a relatively minor invasion into the brain of experimental animal, and could be used advantageously to collect samples for hplc analysis . In our team dialysis fiber based samplers have been developed to measure sampling parameters of microdialysis tubes . In vivo applicability of microdialysis technique combined with in vivo voltammetry for pharmacologycal investigations was checked by application of reserpine and deprenil , selective MAO-B inhibitor [4] to analyze monoamine metabolite concentration changes in the brain of the experimental animal . Method : We have developed microdialysis sampler from Spectrapore HF hollow fiber . Dialysis samplers were tested in vitro in DIAL-CF cell, measuring relative recovery changes as function of flow rate (0 .167 - 10 ul/min) . They were also tested in SCF cell for determination of the response time of in vivo system . The sample collection was done with a perfusion pump, with perfusion rate of 2 ul/min . The hplc system consisted of a Labor-MIM 312 pump, of a thin layer electrochemical detector with a working electrode of glassy carbon . The voltammetric measuring system was controlled by a minicomputer through a fast CAMAC interface, providing different potencial- time curves (as eg . staircase-, double differential pulse voitammetry, chronoamperometry) by software . Adult female cats were anesthetized by nembutal and fixed in a stereotaxic frame . A previously calibrated carbon fiber electrode was inserted into the right striatum, while dialysis sampler was introduced into contralateral striatum at the same coordinate .
1043-6618/92/25110082--02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 25, Supplement 2, 1992
Results : Two different in vitro test systems were employed . The first of was useful for measuring dynamics of in vivo sample collection in an easier way than earlier hplc monitoring system, while the second one was able to perform measurement practically without any dead time . In the SCF cell, the transition time of the sampler was measured . We found 4 .2 min transition time at 10 ul/min flow rate . In the DIAL -CF cell, the relative recoveries were determined for DOPAC, Ascorbic acid, 5-HIAA, HVA and DA solutions to characterize the mass transport through the dialysis membrane . Comparing relative recovery curves of different flow rates (0 .167-10ul/min), values of relative recoveries were found to be between 2 - 95 % . At 2 ul/min, which is the widely used flow rate in brain dialysis methods, the average recovery was about 20 % . When dialysis samplers was implanted into the animal , good correlation was found between voltammetric and respective hplc measurements concerning the concentration changes of electroactive compounds . Increase in monoamine concentrations following reserpine or deprenyl application, indicated also by increase in voltammetric signal (E = + 335 mV), was mostly due to increase in HVA concentration . It is obvious that the dialysis sampling with hplc technique employed simultaneosly can be used to verify the findings of in vivo voltammetric studies . The long term application of dialysis samplers is limited by reactive gliosis which produces a diffusion barrier around the sampler . To reduce this problem we tried to use cytosine arabinoside in the perfusion solution . The measurements of monoamines and their metabolites in different structures could provide good opportunities for the analysis of chemical background neuropharmacological investigations . Ref [1] Ungerstedt U . Measurement of Neurotransmitter Release by Intracranial Dialysis, in : Measurement of Neurotransmitter Release in vivo, Ed . C .A . Marsden, J . Wiley, 1984 :81-105 [2] Johnson RD, Justice JB . Model studies for brain dialysis . Brain Res Bull 1983, 10 :567-571 [3] Church WH, Justice JB . Rapid sampling and determination of extracellular dopamine in vivo . Anal . Chem . 1986, 53 :1649-56 [4] Knoll J, Magyar K . Some puzzling effects of monoamine oxidase inhibitors . Adv Biochem Psychopharmacol 1972, 5 :393-408
83
Pharmacological Research, Vol . 25, Supplement 2, 1992
IN VIVO APPLICABILITY OF BRAIN MICRODIALYSIS IN NEUROPHARMACOLOGICAL INVESTIGATION
J . Tarcali, K . Magyar Department of Pharmacodynamics Semmelweis University of Medicine, Budapest, Hungary Key
words :
in vivo, brain deprenyl, reserpine
microdialysis,
hplc-ec,
Introduction : One of the most promising progress in experimental neuropharmacology in recent years have been achieved in "in vivo" monitoring of extracellular concentrations of neuroactive compounds . Ungerstedt and Justice et al developed flow-trough micro samplers based on single dialysis fiber have been invented for in vivo sample collection (1, 2, 3) . These samplers can be implanted with a relatively minor invasion into the brain of experimental animal, and could be used advantageously to collect samples for hplc analysis . In our team dialysis fiber based samplers have been developed to measure sampling parameters of microdialysis tubes . In vivo applicability of microdialysis technique combined with in vivo voltammetry for pharmacologycal investigations was checked by application of reserpine and deprenil , selective MAO-B inhibitor [4] to analyze monoamine metabolite concentration changes in the brain of the experimental animal . Method : We have developed microdialysis sampler from Spectrapore HF hollow fiber . Dialysis samplers were tested in vitro in DIAL-CF cell, measuring relative recovery changes as function of flow rate (0 .167 - 10 ul/min) . They were also tested in SCF cell for determination of the response time of in vivo system . The sample collection was done with a perfusion pump, with perfusion rate of 2 ul/min . The hplc system consisted of a Labor-MIM 312 pump, of a thin layer electrochemical detector with a working electrode of glassy carbon . The voltammetric measuring system was controlled by a minicomputer through a fast CAMAC interface, providing different potencial- time curves (as eg . staircase-, double differential pulse voitammetry, chronoamperometry) by software . Adult female cats were anesthetized by nembutal and fixed in a stereotaxic frame . A previously calibrated carbon fiber electrode was inserted into the right striatum, while dialysis sampler was introduced into contralateral striatum at the same coordinate .
1043-6618/92/25110082--02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 25, Supplement 2, 1992
Results : Two different in vitro test systems were employed . The first of was useful for measuring dynamics of in vivo sample collection in an easier way than earlier hplc monitoring system, while the second one was able to perform measurement practically without any dead time . In the SCF cell, the transition time of the sampler was measured . We found 4 .2 min transition time at 10 ul/min flow rate . In the DIAL -CF cell, the relative recoveries were determined for DOPAC, Ascorbic acid, 5-HIAA, HVA and DA solutions to characterize the mass transport through the dialysis membrane . Comparing relative recovery curves of different flow rates (0 .167-10ul/min), values of relative recoveries were found to be between 2 - 95 % . At 2 ul/min, which is the widely used flow rate in brain dialysis methods, the average recovery was about 20 % . When dialysis samplers was implanted into the animal , good correlation was found between voltammetric and respective hplc measurements concerning the concentration changes of electroactive compounds . Increase in monoamine concentrations following reserpine or deprenyl application, indicated also by increase in voltammetric signal (E = + 335 mV), was mostly due to increase in HVA concentration . It is obvious that the dialysis sampling with hplc technique employed simultaneosly can be used to verify the findings of in vivo voltammetric studies . The long term application of dialysis samplers is limited by reactive gliosis which produces a diffusion barrier around the sampler . To reduce this problem we tried to use cytosine arabinoside in the perfusion solution . The measurements of monoamines and their metabolites in different structures could provide good opportunities for the analysis of chemical background neuropharmacological investigations . Ref [1] Ungerstedt U . Measurement of Neurotransmitter Release by Intracranial Dialysis, in : Measurement of Neurotransmitter Release in vivo, Ed . C .A . Marsden, J . Wiley, 1984 :81-105 [2] Johnson RD, Justice JB . Model studies for brain dialysis . Brain Res Bull 1983, 10 :567-571 [3] Church WH, Justice JB . Rapid sampling and determination of extracellular dopamine in vivo . Anal . Chem . 1986, 53 :1649-56 [4] Knoll J, Magyar K . Some puzzling effects of monoamine oxidase inhibitors . Adv Biochem Psychopharmacol 1972, 5 :393-408
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