- Email: [email protected]
Measurement of the extracellular H2O2 in the brain by microdialysis
Brain Research Protocols 3 Ž1998. 33–36
Protocol
Measurement of the extracellular H 2 O 2 in the brain by microdialysis Baiping Lei, Naoto Adachi ) , Tatsuru Arai Department of Anesthesiology and Resuscitology, Ehime UniÕersity School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Accepted 7 April 1998
Abstract This paper reports on the protocol for the determination of H 2 O 2 in the brain using in vivo microdialysis coupled with fluorometry of dichlorofluorescin oxidation. We applied this protocol to monitor changes in the concentration of H 2 O 2 in the brain, in vivo, during ischemia and reperfusion. Using this method, changes in the level of H 2 O 2 in the brain during ischemia and reperfusion were effectively determined. The present protocol provides a novel tool to study the production of reactive oxygen species in the brain. q 1998 Elsevier Science B.V. All rights reserved. Themes: Disorders of the nervous system Topics: Ischemia
Keywords: Hydrogen peroxide; Dichlorofluorescin; Microdialysis; Cerebral ischemia; Hippocampus; Mongolian gerbil
1. Type of research The present protocol may be applied to any types of research that requires in vivo monitoring of the H 2 O 2 generation in the brain by microdialysis. Since the extracellular H 2 O 2 measured by brain microdialysis reflects the intracellular reactive oxygen species ŽROS., this protocol is useful to monitor the production of ROS in the brain. Possible applications are: Ø Study on the temporal pattern of the H 2 O 2 production in cerebral ischemia and reperfusion. Ø Measurements of H 2 O 2 in the in vivo brain after various treatments and experimental manipulations. Ø Studies on relationships between neurotransmitters and the H 2 O 2 production in various pathological conditions.
2. Time required Ø In vivo microdialysis: this step is dependent on the type of experiment. Ø Whole protocol: the time required for microdialysis plus 1.5 h. )
Corresponding author. Fax: q81-89-960-5386.
3. Materials Special equipment Ø Brain stereotaxic apparatus ŽDavid Kopf Instruments, Tujunga, CA, USA.. Ø Microdialysis probe guide cannula ŽA-I-8, Eicom, Kyoto, Japan.. Ø Microdialysis probe Ž1 mm long, o.d. 0.22 mm, molecular weight cut off at 50,000; A-I-8-01, Eicom.. Ø Microinjection pump ŽModel 210, Kd Scientific, USA.. Ø Thermometer ŽDT-300, Inter Medical, Nagano, Japan.. Ø Heating lamp ŽModel TGHM, Olympus, Tokyo, Japan.. Ø Spectrofluorometer ŽFP-777, JASCO, Tokyo, Japan.. Ø Spectrophotometer ŽU-3000, Hitachi, Tokyo, Japan.. Chemicals and reagents Ø 2X ,7X-dichlorofluorescin diacetate ŽDCFH-DA. from Molecular Probes ŽEugene, OR, USA.. Ø Horseradish peroxidase ŽEC 1.11.1.7, type VI, from Horseradish; 318 purpurogallin unitsrmg solid. from Sigma ŽSt. Louis, MO, USA.. Ø Catalase ŽEC 1.11.1.6, from bovine liver, 0.4 ml, 140 mg proteinrml, 41,000 unitsrmg protein. from Sigma. Ø H 2 O 2 Ž30 WrV%., ethanol Ž99.5 VrV%., 0.01 M
1385-299Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 1 8 - X
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
34
NaOH, ZnSO4 P 7H 2 O, Na 2 HPO4 P 12H 2 O, and NaH 2 PO4 from Wako Pure Chemical Industries ŽOsaka, Japan..
4. Detailed procedure
membrane is located in the CA1 field w7x. Perfuse the probe with Ringer’s solution at a constant flow rate of 2 m lrmin with a microinjection pump. Ø After a stabilization period of 90 min, collect dialysates every 10 min into microtubes on ice and store them at y808C. Ø Verify histologically the location of the microdialysis probe in each animal at the end of the experiment.
4.1. In ÕiÕo microdialysis 4.1.1. Animals Male Mongolian gerbils Ž60–80 g. ŽSeiwa Experimental Animals, Fukuoka, Japan. were housed in a room controlled at 23 " 28C with controlled lighting conditions Ž12 h lightr12 h dark.. Food and water were provided ad libitum. The gerbil is unique in having an incomplete circulus of Willis, which allows transient global ischemia to be achieved by occluding bilateral common carotid arteries. Global ischemia for 5 min causes a selective pattern of neurodegeneration in the hippocampal CA1 region w9x. This model is often used for studying the mechanisms of ischemic brain damage and the effects of neuroprotective agents.
4.1.4. Transient forebrain ischemia Pull the threads around both common carotid arteries with 8-g weights. Following 5 min of ischemia, cut the threads to restore the blood flow w7x. Likewise, a sham-operated group without ischemia should be studied. 4.1.5. In Õitro recoÕery rate Ø To determine the recovery rate of H 2 O 2 across the membrane, place the microdialysis probe in a Ringer’s solution containing 10 m M H 2 O 2 in a 378C water bath. Perfuse Ringer’s solution through the probe at a rate of 2 m lrmin. Ø Discard the first 15-min perfusate. Collect three fractions every 10 min for analysis of H 2 O 2 content. Ø Calculate the ratio by dividing the concentration of the perfusate by the concentration outside the dialysis membrane. It should be noted that the in vitro recovery cannot be directly extrapolated to in vivo samples. It is better to present the data as the directly measured concentrations in the dialysate. To determine actual concentrations in the extracellular fluid, a more elaborate in vivo procedure is necessary w4x.
4.1.2. Surgical preparation Ø Anesthetize and maintain the gerbil with 3% halothane in a balanced 50% nitrous oxide with a face mask. Ø Make a median neck incision and expose both common carotid arteries. Separate them carefully from adjacent nerves and tissues. Loop a silk thread Ž4-0. around each common carotid artery. Ø Place the animal in a stereotaxic apparatus and expose the skull with a median scalp incision. Drill two small burr holes in the skull for insertion of a temperature probe and a microdialysis probe. Incise the dura with the tip of a needle w1,7x. Ø Insert a thermocouple needle-probe in the right brain through one burr hole drilled in the skull: the tip is positioned ; 3.5 mm anterior and 2 mm lateral to the bregma and 2 mm below the brain surface. It is necessary to monitor the brain temperature during the whole experiment, because changes in brain temperature will exert influences on ischemic brain damage. Ø Implant a guide cannula in the left brain through the other burr hole drilled in the cranium Ž2 mm posterior and 2 mm lateral to the bregma and 1.5 mm below the brain surface.. Ø Maintain anesthesia with 2% halothane. Keep brain and rectal temperatures at 37–37.58C with a heating lamp throughout the experiment.
4.2.1. Preparation of stock solutions Stock solutions should be prepared prior to the assay and stored. a. Dissolve DCFH-DA in ethanol at the concentration of 10 mM and keep under y808C until used. Stock solutions Ž40 m l each. should be made on a daily basis. All experiments should be performed using a single batch of DCFH-DA. b. Dissolve horseradish peroxidase with 2.5 mM sodium phosphate buffer ŽpH 7.2. at 2 mgrml. Centrifuge at 3000 = g for 10 min Ž48C.. Freeze small aliquots Ž25 m l each. at y208C until used. c. Prepare an H 2 O 2 stock solution by diluting 30% H 2 O 2 with distilled deionized water. Standardize the stock solution Ž10 mM. by using 43.6 Žmolrl.y1 cmy1 as the molar extinction coefficient at 240 nm. d. Prepare sodium phosphate buffer Ž2.5 mM, pH 7.20. biweekly and store at 48C.
4.1.3. Microdialysis procedure Ø Insert a microdialysis probe into the left hippocampus through the guide cannula. The portion of dialysis
4.2.2. Preparation of working solutions Working solutions should be made each day. a. Prepare DCFH solution by mixing 40 m l of 10 mM DCFH-DA in ethanol with 100 m l of 0.01 M NaOH. This
4.2. Determination of H2 O2 in microdialysis samples
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
deesterification of DCFH-DA proceeds at room temperature for 15 min. Add the mixture to 200 ml of 2.5 mM sodium phosphate buffer ŽpH 7.20. containing 8 mg of ZnSO4 P 7H 2 O Ž40 m grml. and 4 m l of horseradish peroxidase stock solution Ž40 ngrml.. The final concentration for DCFH is about 2 m M. Keep this DCFH working solution on ice in the dark until used. Note that this solution should be used within a few hours to decrease the blank value, although autofluorescence is always less than 30 arbitrary fluorescence units per hour. b. Prepare H 2 O 2 standard solutions Ž0–5 m M. by diluting the stock standard solution Ž10 m M. with distilled deionized water. c. Prepare catalase solution by adding 4 m l of catalase to 20 ml cold phosphate buffer. The final concentration of catalase is about 1000 Urml. Keep it on ice. 4.2.3. Determination of H2 O2 The amount of H 2 O 2 in dialysates is determined by the method of Keston and Brandt w5x with a modification. a. Add a 10-m l aliquot of the sample into a 10 = 75 mm test tube containing 50 m l of 2.5 mM sodium phosphate buffer ŽpH 7.20.. Initiate the reaction by adding 500 m l of DCFH working solution. Incubate the tube for 15 min at 378C in a water bath in the dark. Cool the tube for 2 min in water Žroom temperature.. Measure the fluorescence intensity at an excitation wavelength of 508 nm and an emission wavelength of 538 nm with a model FP-777 spectrofluorometer. b. Add another 10-m l aliquot of the sample to a test tube containing 50 m l of catalase solution. After 10-min incubation at room temperature, add 500 m l of DCFH working solution to the test tube to initiate the reaction. Determine the fluorescence intensity as described above. c. Treat the blank tube ŽRinger’s solution. in an identical manner as the sample.
35
Fig. 2. Changes in the H 2 O 2 concentration in dialysates in the gerbil hippocampus subjected to transient forebrain ischemia for 5 min. Each value represents the mean"SD for five animals. The solid rectangle designates the duration of ischemia. Data were subjected to ANOVA followed by Sheffe’s ´ test. ) p- 0.01 compared with the corresponding values in the sham-operated group.
d. Generate a standard curve with 0–5 m M of H 2 O 2 as described above. Note, this must be done in parallel with the samples throughout the assay in each experiment. e. Correct the fluorescence intensity by subtracting the value of the sample treated with catalase from that without catalase. Calculate the concentration of H 2 O 2 in the sample from the H 2 O 2 standard curve. 5. Results As shown in Fig. 1, the fluorescence intensity is linear for a wide range of the concentration. The amount of H 2 O 2 between 1 and 50 pmol can be determined without a modification. This range is very suitable for the determination of H 2 O 2 in microdialysis samples. The in vitro recovery rate of H 2 O 2 through the dialysis membrane at 378C was about 7.0 " 0.5% Žmean " SD for five probes.. Fig. 2 shows changes in the H 2 O 2 concentration in dialysates in the gerbil hippocampus during ischemia and reperfusion. As previously reported, the basal concentrations of H 2 O 2 in dialysates prior to ischemia was about 1–2 m M w7x. In the sham-operated group, there was a slight decrease in the H 2 O 2 level throughout the experimental period. In the ischemic group, transient forebrain ischemia for 5 min produced a marked and rapid increase in the hippocampal H 2 O 2 immediately after the start of ischemia, and the elevation continued until 30 min after reperfusion. 6. Discussion
Fig. 1. A standard curve of H 2 O 2 . Fluorescence intensity from each amount of H 2 O 2 was determined after subtracting the blank value. The data were obtained from five independent measurements and are expressed as the mean"SD.
6.1. Troubleshooting A. The sensitivity of this fluorescent dichlorofluorescin assay for the detection of H 2 O 2 is dependent on the
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
36
concentration of horseradish peroxidase in the reaction mixture. Horseradish peroxidase can oxidize DCFH in the absence of H 2 O 2 w6x. The oxidative action of horseradish peroxidase contributes to the main portion of the blank fluorescence w5x. Under the condition described, the fluorescence intensity of the blank ranges from 300 to 600 fluorescent units. One pmol of H 2 O 2 normally yields a fluorescence intensity of more than 50 fluorescent units at a gain setting of very low. The concentration of DCFH in the reactive mixture also affects the sensitivity and the range of the assay. The sensitivity and the range of the assay can be adjusted by changing the concentrations of horseradish peroxidase and DCFH. The value of the blank may be decreased by shortening the incubation time. It is recommended to determine the sensitivity and the range of the assay before analyzing samples. B. Because several reactive intermediates of ROS as well as H 2 O 2 causes DCFH oxidation w6x, samples should be treated with catalase to exclude the element besides H 2 O 2 . The fluorescence intensity of catalase-treated dialysates is 20%–40% of that of dialysates without catalase treatment, and the ratio differs in the portion of the brain.
C. Determination of H 2 O 2 . Ø Deesterify DCFH-DA. Ø Prepare the solution containing DCFH, horseradish peroxidase and ZnSO4 P 7H 2 O. Ø Prepare the catalase solution. Ø Prepare H 2 O 2 standard solution. Ø Add an aliquot of the sample to the test tube with and without catalase. Ø Add DCFH solution to the test tube and incubate the tube at 378C for 15 min. Ø Measure the fluorescence intensity.
8. Essential literature references References w4–7x.
Acknowledgements We thank Dr. Likun Han ŽSecond Department of Medical Biochemistry. for his useful technical assistance.
6.2. Application of the protocol The present report describes a simple and convenient method to assay the extracellular H 2 O 2 . We applied the protocol to study the effect of hypothermia on the H 2 O 2 production during ischemia and reperfusion w7x, and the effect of dopamine depletion on the H 2 O 2 production in the rat striatum following transient middle cerebral artery occlusion w8x. Microdialysis can effectively measure changes in the extracellular level of H 2 O 2 in the brain during ischemia and reperfusion w4x. Because H 2 O 2 freely crosses biological membranes and the rate of its production is closely related to those of superoxide anions and hydroxyl radicals w3x, it is presumed that the extracellular H 2 O 2 measured by brain microdialysis reflects the intracellular ROS. Thus, the measurement of the extracellular level of H 2 O 2 by microdialysis is thought to be a reliable method to monitor ROS. The pattern of changes in the H 2 O 2 concentration in the in vivo brain during ischemia and reperfusion in our studies is in good agreement with those in superoxide anions and hydroxyl radicals examined by the spin-trapping or the salicylate trapping method w2,10x. The present protocol provides a novel tool to study the ROS production in the in vivo brain, which may be advantageous over the existing methods that require trapping agents. 7. Quick procedure A. Sampling with in vivo microdialysis: of your choice. B. Preparation of DCFH-DA, horseradish peroxidase and H 2 O 2 stock solutions.
References w1x N. Adachi, Y. Itoh, R. Oishi, K. Saeki, Direct evidence for increased continuous histamine release in the striatum of conscious freely moving rats produced by middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 12 Ž1992. 477–483. w2x M.Y.-T. Globus, R. Busto, B. Lin, H. Schnippering, M.D. Ginsberg, Detection of free radical activity during transient global ischemia and recirculation: effects of intraischemic brain temperature modulation, J. Neurochem. 65 Ž1995. 1250–1256. w3x B. Halliwell, J.M.C. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186 Ž1990. 1–85. w4x P.A. Hyslop, Z. Zhang, D.V. Pearson, L.A. Phebus, Measurement of striatal H 2 O 2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H 2 O 2 in vitro, Brain Res. 671 Ž1995. 181–186. w5x A.S. Keston, R. Brandt, The fluorometric analysis of ultramicro quantities of hydrogen peroxide, Anal. Biochem. 11 Ž1965. 1–5. w6x C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe X X 2 ,7 -dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress, Chem. Res. Toxicol. 5 Ž1992. 227– 231. w7x B. Lei, N. Adachi, T. Arai, The effect of hypothermia on H 2 O 2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus, Neurosci. Lett. 222 Ž1997. 91–94. w8x B. Lei, N. Adachi, T. Nagaro, T. Arai, The effect of dopamine depletion on the H 2 O 2 production in the rat striatum following transient middle cerebral artery occlusion, Brain Res. 764 Ž1997. 299–302. w9x S. Levine, H. Payan, Effects of ischemia and other procedures on the brain and retina of the gerbil Ž Meriones unguiculatus., Exp. Neurol. 16 Ž1966. 255–262. w10x I. Zini, A. Tomasi, R. Grimaldi, V. Vannini, L.F. Agnati, Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis, Neurosci. Lett. 138 Ž1992. 279–282.
Protocol
Measurement of the extracellular H 2 O 2 in the brain by microdialysis Baiping Lei, Naoto Adachi ) , Tatsuru Arai Department of Anesthesiology and Resuscitology, Ehime UniÕersity School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan Accepted 7 April 1998
Abstract This paper reports on the protocol for the determination of H 2 O 2 in the brain using in vivo microdialysis coupled with fluorometry of dichlorofluorescin oxidation. We applied this protocol to monitor changes in the concentration of H 2 O 2 in the brain, in vivo, during ischemia and reperfusion. Using this method, changes in the level of H 2 O 2 in the brain during ischemia and reperfusion were effectively determined. The present protocol provides a novel tool to study the production of reactive oxygen species in the brain. q 1998 Elsevier Science B.V. All rights reserved. Themes: Disorders of the nervous system Topics: Ischemia
Keywords: Hydrogen peroxide; Dichlorofluorescin; Microdialysis; Cerebral ischemia; Hippocampus; Mongolian gerbil
1. Type of research The present protocol may be applied to any types of research that requires in vivo monitoring of the H 2 O 2 generation in the brain by microdialysis. Since the extracellular H 2 O 2 measured by brain microdialysis reflects the intracellular reactive oxygen species ŽROS., this protocol is useful to monitor the production of ROS in the brain. Possible applications are: Ø Study on the temporal pattern of the H 2 O 2 production in cerebral ischemia and reperfusion. Ø Measurements of H 2 O 2 in the in vivo brain after various treatments and experimental manipulations. Ø Studies on relationships between neurotransmitters and the H 2 O 2 production in various pathological conditions.
2. Time required Ø In vivo microdialysis: this step is dependent on the type of experiment. Ø Whole protocol: the time required for microdialysis plus 1.5 h. )
Corresponding author. Fax: q81-89-960-5386.
3. Materials Special equipment Ø Brain stereotaxic apparatus ŽDavid Kopf Instruments, Tujunga, CA, USA.. Ø Microdialysis probe guide cannula ŽA-I-8, Eicom, Kyoto, Japan.. Ø Microdialysis probe Ž1 mm long, o.d. 0.22 mm, molecular weight cut off at 50,000; A-I-8-01, Eicom.. Ø Microinjection pump ŽModel 210, Kd Scientific, USA.. Ø Thermometer ŽDT-300, Inter Medical, Nagano, Japan.. Ø Heating lamp ŽModel TGHM, Olympus, Tokyo, Japan.. Ø Spectrofluorometer ŽFP-777, JASCO, Tokyo, Japan.. Ø Spectrophotometer ŽU-3000, Hitachi, Tokyo, Japan.. Chemicals and reagents Ø 2X ,7X-dichlorofluorescin diacetate ŽDCFH-DA. from Molecular Probes ŽEugene, OR, USA.. Ø Horseradish peroxidase ŽEC 1.11.1.7, type VI, from Horseradish; 318 purpurogallin unitsrmg solid. from Sigma ŽSt. Louis, MO, USA.. Ø Catalase ŽEC 1.11.1.6, from bovine liver, 0.4 ml, 140 mg proteinrml, 41,000 unitsrmg protein. from Sigma. Ø H 2 O 2 Ž30 WrV%., ethanol Ž99.5 VrV%., 0.01 M
1385-299Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 1 8 - X
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
34
NaOH, ZnSO4 P 7H 2 O, Na 2 HPO4 P 12H 2 O, and NaH 2 PO4 from Wako Pure Chemical Industries ŽOsaka, Japan..
4. Detailed procedure
membrane is located in the CA1 field w7x. Perfuse the probe with Ringer’s solution at a constant flow rate of 2 m lrmin with a microinjection pump. Ø After a stabilization period of 90 min, collect dialysates every 10 min into microtubes on ice and store them at y808C. Ø Verify histologically the location of the microdialysis probe in each animal at the end of the experiment.
4.1. In ÕiÕo microdialysis 4.1.1. Animals Male Mongolian gerbils Ž60–80 g. ŽSeiwa Experimental Animals, Fukuoka, Japan. were housed in a room controlled at 23 " 28C with controlled lighting conditions Ž12 h lightr12 h dark.. Food and water were provided ad libitum. The gerbil is unique in having an incomplete circulus of Willis, which allows transient global ischemia to be achieved by occluding bilateral common carotid arteries. Global ischemia for 5 min causes a selective pattern of neurodegeneration in the hippocampal CA1 region w9x. This model is often used for studying the mechanisms of ischemic brain damage and the effects of neuroprotective agents.
4.1.4. Transient forebrain ischemia Pull the threads around both common carotid arteries with 8-g weights. Following 5 min of ischemia, cut the threads to restore the blood flow w7x. Likewise, a sham-operated group without ischemia should be studied. 4.1.5. In Õitro recoÕery rate Ø To determine the recovery rate of H 2 O 2 across the membrane, place the microdialysis probe in a Ringer’s solution containing 10 m M H 2 O 2 in a 378C water bath. Perfuse Ringer’s solution through the probe at a rate of 2 m lrmin. Ø Discard the first 15-min perfusate. Collect three fractions every 10 min for analysis of H 2 O 2 content. Ø Calculate the ratio by dividing the concentration of the perfusate by the concentration outside the dialysis membrane. It should be noted that the in vitro recovery cannot be directly extrapolated to in vivo samples. It is better to present the data as the directly measured concentrations in the dialysate. To determine actual concentrations in the extracellular fluid, a more elaborate in vivo procedure is necessary w4x.
4.1.2. Surgical preparation Ø Anesthetize and maintain the gerbil with 3% halothane in a balanced 50% nitrous oxide with a face mask. Ø Make a median neck incision and expose both common carotid arteries. Separate them carefully from adjacent nerves and tissues. Loop a silk thread Ž4-0. around each common carotid artery. Ø Place the animal in a stereotaxic apparatus and expose the skull with a median scalp incision. Drill two small burr holes in the skull for insertion of a temperature probe and a microdialysis probe. Incise the dura with the tip of a needle w1,7x. Ø Insert a thermocouple needle-probe in the right brain through one burr hole drilled in the skull: the tip is positioned ; 3.5 mm anterior and 2 mm lateral to the bregma and 2 mm below the brain surface. It is necessary to monitor the brain temperature during the whole experiment, because changes in brain temperature will exert influences on ischemic brain damage. Ø Implant a guide cannula in the left brain through the other burr hole drilled in the cranium Ž2 mm posterior and 2 mm lateral to the bregma and 1.5 mm below the brain surface.. Ø Maintain anesthesia with 2% halothane. Keep brain and rectal temperatures at 37–37.58C with a heating lamp throughout the experiment.
4.2.1. Preparation of stock solutions Stock solutions should be prepared prior to the assay and stored. a. Dissolve DCFH-DA in ethanol at the concentration of 10 mM and keep under y808C until used. Stock solutions Ž40 m l each. should be made on a daily basis. All experiments should be performed using a single batch of DCFH-DA. b. Dissolve horseradish peroxidase with 2.5 mM sodium phosphate buffer ŽpH 7.2. at 2 mgrml. Centrifuge at 3000 = g for 10 min Ž48C.. Freeze small aliquots Ž25 m l each. at y208C until used. c. Prepare an H 2 O 2 stock solution by diluting 30% H 2 O 2 with distilled deionized water. Standardize the stock solution Ž10 mM. by using 43.6 Žmolrl.y1 cmy1 as the molar extinction coefficient at 240 nm. d. Prepare sodium phosphate buffer Ž2.5 mM, pH 7.20. biweekly and store at 48C.
4.1.3. Microdialysis procedure Ø Insert a microdialysis probe into the left hippocampus through the guide cannula. The portion of dialysis
4.2.2. Preparation of working solutions Working solutions should be made each day. a. Prepare DCFH solution by mixing 40 m l of 10 mM DCFH-DA in ethanol with 100 m l of 0.01 M NaOH. This
4.2. Determination of H2 O2 in microdialysis samples
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
deesterification of DCFH-DA proceeds at room temperature for 15 min. Add the mixture to 200 ml of 2.5 mM sodium phosphate buffer ŽpH 7.20. containing 8 mg of ZnSO4 P 7H 2 O Ž40 m grml. and 4 m l of horseradish peroxidase stock solution Ž40 ngrml.. The final concentration for DCFH is about 2 m M. Keep this DCFH working solution on ice in the dark until used. Note that this solution should be used within a few hours to decrease the blank value, although autofluorescence is always less than 30 arbitrary fluorescence units per hour. b. Prepare H 2 O 2 standard solutions Ž0–5 m M. by diluting the stock standard solution Ž10 m M. with distilled deionized water. c. Prepare catalase solution by adding 4 m l of catalase to 20 ml cold phosphate buffer. The final concentration of catalase is about 1000 Urml. Keep it on ice. 4.2.3. Determination of H2 O2 The amount of H 2 O 2 in dialysates is determined by the method of Keston and Brandt w5x with a modification. a. Add a 10-m l aliquot of the sample into a 10 = 75 mm test tube containing 50 m l of 2.5 mM sodium phosphate buffer ŽpH 7.20.. Initiate the reaction by adding 500 m l of DCFH working solution. Incubate the tube for 15 min at 378C in a water bath in the dark. Cool the tube for 2 min in water Žroom temperature.. Measure the fluorescence intensity at an excitation wavelength of 508 nm and an emission wavelength of 538 nm with a model FP-777 spectrofluorometer. b. Add another 10-m l aliquot of the sample to a test tube containing 50 m l of catalase solution. After 10-min incubation at room temperature, add 500 m l of DCFH working solution to the test tube to initiate the reaction. Determine the fluorescence intensity as described above. c. Treat the blank tube ŽRinger’s solution. in an identical manner as the sample.
35
Fig. 2. Changes in the H 2 O 2 concentration in dialysates in the gerbil hippocampus subjected to transient forebrain ischemia for 5 min. Each value represents the mean"SD for five animals. The solid rectangle designates the duration of ischemia. Data were subjected to ANOVA followed by Sheffe’s ´ test. ) p- 0.01 compared with the corresponding values in the sham-operated group.
d. Generate a standard curve with 0–5 m M of H 2 O 2 as described above. Note, this must be done in parallel with the samples throughout the assay in each experiment. e. Correct the fluorescence intensity by subtracting the value of the sample treated with catalase from that without catalase. Calculate the concentration of H 2 O 2 in the sample from the H 2 O 2 standard curve. 5. Results As shown in Fig. 1, the fluorescence intensity is linear for a wide range of the concentration. The amount of H 2 O 2 between 1 and 50 pmol can be determined without a modification. This range is very suitable for the determination of H 2 O 2 in microdialysis samples. The in vitro recovery rate of H 2 O 2 through the dialysis membrane at 378C was about 7.0 " 0.5% Žmean " SD for five probes.. Fig. 2 shows changes in the H 2 O 2 concentration in dialysates in the gerbil hippocampus during ischemia and reperfusion. As previously reported, the basal concentrations of H 2 O 2 in dialysates prior to ischemia was about 1–2 m M w7x. In the sham-operated group, there was a slight decrease in the H 2 O 2 level throughout the experimental period. In the ischemic group, transient forebrain ischemia for 5 min produced a marked and rapid increase in the hippocampal H 2 O 2 immediately after the start of ischemia, and the elevation continued until 30 min after reperfusion. 6. Discussion
Fig. 1. A standard curve of H 2 O 2 . Fluorescence intensity from each amount of H 2 O 2 was determined after subtracting the blank value. The data were obtained from five independent measurements and are expressed as the mean"SD.
6.1. Troubleshooting A. The sensitivity of this fluorescent dichlorofluorescin assay for the detection of H 2 O 2 is dependent on the
B. Lei et al.r Brain Research Protocols 3 (1998) 33–36
36
concentration of horseradish peroxidase in the reaction mixture. Horseradish peroxidase can oxidize DCFH in the absence of H 2 O 2 w6x. The oxidative action of horseradish peroxidase contributes to the main portion of the blank fluorescence w5x. Under the condition described, the fluorescence intensity of the blank ranges from 300 to 600 fluorescent units. One pmol of H 2 O 2 normally yields a fluorescence intensity of more than 50 fluorescent units at a gain setting of very low. The concentration of DCFH in the reactive mixture also affects the sensitivity and the range of the assay. The sensitivity and the range of the assay can be adjusted by changing the concentrations of horseradish peroxidase and DCFH. The value of the blank may be decreased by shortening the incubation time. It is recommended to determine the sensitivity and the range of the assay before analyzing samples. B. Because several reactive intermediates of ROS as well as H 2 O 2 causes DCFH oxidation w6x, samples should be treated with catalase to exclude the element besides H 2 O 2 . The fluorescence intensity of catalase-treated dialysates is 20%–40% of that of dialysates without catalase treatment, and the ratio differs in the portion of the brain.
C. Determination of H 2 O 2 . Ø Deesterify DCFH-DA. Ø Prepare the solution containing DCFH, horseradish peroxidase and ZnSO4 P 7H 2 O. Ø Prepare the catalase solution. Ø Prepare H 2 O 2 standard solution. Ø Add an aliquot of the sample to the test tube with and without catalase. Ø Add DCFH solution to the test tube and incubate the tube at 378C for 15 min. Ø Measure the fluorescence intensity.
8. Essential literature references References w4–7x.
Acknowledgements We thank Dr. Likun Han ŽSecond Department of Medical Biochemistry. for his useful technical assistance.
6.2. Application of the protocol The present report describes a simple and convenient method to assay the extracellular H 2 O 2 . We applied the protocol to study the effect of hypothermia on the H 2 O 2 production during ischemia and reperfusion w7x, and the effect of dopamine depletion on the H 2 O 2 production in the rat striatum following transient middle cerebral artery occlusion w8x. Microdialysis can effectively measure changes in the extracellular level of H 2 O 2 in the brain during ischemia and reperfusion w4x. Because H 2 O 2 freely crosses biological membranes and the rate of its production is closely related to those of superoxide anions and hydroxyl radicals w3x, it is presumed that the extracellular H 2 O 2 measured by brain microdialysis reflects the intracellular ROS. Thus, the measurement of the extracellular level of H 2 O 2 by microdialysis is thought to be a reliable method to monitor ROS. The pattern of changes in the H 2 O 2 concentration in the in vivo brain during ischemia and reperfusion in our studies is in good agreement with those in superoxide anions and hydroxyl radicals examined by the spin-trapping or the salicylate trapping method w2,10x. The present protocol provides a novel tool to study the ROS production in the in vivo brain, which may be advantageous over the existing methods that require trapping agents. 7. Quick procedure A. Sampling with in vivo microdialysis: of your choice. B. Preparation of DCFH-DA, horseradish peroxidase and H 2 O 2 stock solutions.
References w1x N. Adachi, Y. Itoh, R. Oishi, K. Saeki, Direct evidence for increased continuous histamine release in the striatum of conscious freely moving rats produced by middle cerebral artery occlusion, J. Cereb. Blood Flow Metab. 12 Ž1992. 477–483. w2x M.Y.-T. Globus, R. Busto, B. Lin, H. Schnippering, M.D. Ginsberg, Detection of free radical activity during transient global ischemia and recirculation: effects of intraischemic brain temperature modulation, J. Neurochem. 65 Ž1995. 1250–1256. w3x B. Halliwell, J.M.C. Gutteridge, Role of free radicals and catalytic metal ions in human disease: an overview, Methods Enzymol. 186 Ž1990. 1–85. w4x P.A. Hyslop, Z. Zhang, D.V. Pearson, L.A. Phebus, Measurement of striatal H 2 O 2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H 2 O 2 in vitro, Brain Res. 671 Ž1995. 181–186. w5x A.S. Keston, R. Brandt, The fluorometric analysis of ultramicro quantities of hydrogen peroxide, Anal. Biochem. 11 Ž1965. 1–5. w6x C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe X X 2 ,7 -dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress, Chem. Res. Toxicol. 5 Ž1992. 227– 231. w7x B. Lei, N. Adachi, T. Arai, The effect of hypothermia on H 2 O 2 production during ischemia and reperfusion: a microdialysis study in the gerbil hippocampus, Neurosci. Lett. 222 Ž1997. 91–94. w8x B. Lei, N. Adachi, T. Nagaro, T. Arai, The effect of dopamine depletion on the H 2 O 2 production in the rat striatum following transient middle cerebral artery occlusion, Brain Res. 764 Ž1997. 299–302. w9x S. Levine, H. Payan, Effects of ischemia and other procedures on the brain and retina of the gerbil Ž Meriones unguiculatus., Exp. Neurol. 16 Ž1966. 255–262. w10x I. Zini, A. Tomasi, R. Grimaldi, V. Vannini, L.F. Agnati, Detection of free radicals during brain ischemia and reperfusion by spin trapping and microdialysis, Neurosci. Lett. 138 Ž1992. 279–282.