In Vivo Detection of Superoxide Anion Production by

Journal of Cerebral Blood Flow and Metabolism 15:242-247 © 1995 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press. Ltd .• New York

In Vivo Detection of Superoxide Anion Production by the Brain Using a Cytochrome c Electrode *IIRoderic H. Fabian, tDouglas S. DeWitt, and *:j:§�Thomas A. Kent Departments of*Neurology, tAnesthesiology, tPsychiatry and Behavioral Sciences, and §Pharmacology and Toxicology and �Marine Biomedical Institute, University of Texas Medical Branch at Galveston, Galveston, Texas, U.S.A.

Summary: A cytochrome c-coated platinized carbon electrode was utilized to detect superoxide generated by the brain during hypoxialhypercarbia, focal ischemia, and reperfusion and following fluid percussion brain injury with and without hemorrhagic hypotension and reperfu­ sion in the rat. All three of these forms of brain injury

were associated with an increase in the superoxide signal. The cytochrome c electrode proved to be sensitive and responsive enough for minute-by-minute measurement of superoxide generation by brain tissue. Key Words: Brain-Cytochrome c-Electrode-Superoxide anion.

Oxygen-derived free radicals such as the super­ oxide anion and the hydroxyl radical are known to be involved in many biological processes. Superox­ ide, in particular, is produced by neutrophils and mononuclear phagocytes as part of their antimicro­ bial armamentarium and may be produced in excess by certain metabolic processes, such as porphyrin and arachidonic acid metabolism (Braughler and Hall, 1989). In some situations, the production of superoxide may become excessive, leading to sec­ ondary tissue damage. This is thought to occur in oxygen toxicity, in ischemia, and in other forms of tissue injury (U yama et aI., 1990; Cazevieille et aI., 1993). The study of superoxide and other free rad­ icals has been hampered by the difficulties involved in detecting and measuring them. Previous investi­ gators have measured the production of various free radicals using nitroblue tetrazolium reduction (Kon­ tos et aI., 1984; Armstead et aI., 1989), chemilumi-

nescence (Imaizumi et aI., 1984), electron spin res­ onance spectroscopy (Tominaga et aI., 1985; Lai et aI., 1986), and salicylate trapping (Cao et aI., 1988; Globus et aI., 1994). Recently, McNeil et al. ( 1992) reported using cytochrome c electrodes for the elec­ trochemical measurement of superoxide in vitro. In this report, we describe the use of cytochrome c-coated (CC) electrodes for reagentless, in vivo measurement of superoxide production by the brain. We applied this technique to measure super­ oxide production by the brain during asphyxia, ce­ rebral trauma and hypotension, and focal cerebral ischemia with reperfusion. METHODS Calibration of electrodes using xanthine-xanthine oxidase system in vitro

Cytochrome c electrodes were prepared using a modi­ fication of the method of McNeil et al. ( 1992). Electrodes were constructed of platinized carbon electrode (PACE) material (EFCG S type material; E-Tek, Natick, MA, U.S.A.), which was the generous gift of Dr. Calum Mc­ Neil. PACE material has been found to adsorb proteins with great efficiency, providing for very sensitive enzyme electrodes. Nine-square-millimeter pieces of PACE ma­ terial were placed in absolute ethanol for 12 h and trans­ ferred to 1 % sodium dodecyl sulfate solution fQr several hours. The material was washed in four changes of dis­ tilled water before being transferred to phosphate­ buffered saline (PBS), pH 7.4, at 4°C overnight. The ma­ terial was then placed in 10 mg/ml cytochrome c (Sigma)

Received March 8, 1994; final revision received June 23, 1994; accepted June 28, 1994. Address correspondence and reprint requests to Dr. R. H. Fabian at Department of Neurology, University of Texas Med­ ical Branch, 301 University Blvd. , Galveston, TX 77555-0539, U. S. A. Abbreviations used: CC, cytochrome c-coated; HBSS, Hanks' Balanced Salt Solution; PACE, platinized carbon electrode; PBS, phosphate-buffered saline; SCE, saturated mercury­ calamine electrode; SOD, superoxide dismutase; TBI, traumatic brain injury.

242

CYTOCHROME in PBS at 4°C for 24 h. The CC-PACE material was rinsed and stored in PBS at 4°C until use. Electrodes were pre­ pared by fixing 2- or 3-mm squares of PACE material to thin platinum wire loops. The resulting electrode assem­ bly was calibrated for sensitivity to superoxide using the xanthine-xanthine oxidase system. Solutions of 0.5 mM xanthine in Hanks' Balanced Salt Solution (HBSS) were kept at 2YC and agitated in 15-ml electrochemical cells. Current measurements were made using an E' -Chern DCV-5 potentiostat (BAS, West Lafayette, IN, U.S.A.) with the PACE versus a platinum wire counterelectrode with a constant applied voltage of 0.05 V relative to a saturated mercury-calamine electrode (SCE). Five to 500 fLg of xanthine oxidase (Sigma Chemical Co.) in HBSS was added and the resulting current change recorded for a period of several minutes. The sensitivity of the CC­ PACE was taken as the reciprocal of the maximum cur­ rent change recorded relative to the predicted rate of su­ peroxide production per unit volume of buffer and the surface area of the electrode. Response of cytochrome c electrode to nitric oxide and hydroxyl radical

To determine whether or not the cytochrome c elec­ trode would produce a current change on exposure to nitric oxide, sodium nitroprusside, 0. 1- 100 mM, was added to the electrochemical cell containing HBSS and the current was recorded for a period of several minutes as previously. To determine whether or not the electrode would produce a current change on exposure to hydroxyl radical, ferrous sulfate, I-50 fLM, was added to the elec­ trochemical cell containing HBSS with 0.5 mM hydrogen peroxide, and the current was recorded for a period of several minutes as described. Superoxide production with hypoxemia/hypercarhia

All experimental protocols were approved by the Ani­ mal Care and Use Committee of the University of Texas Medical Branch. Adult Sprague-Dawley rats of either sex (�450 g) were anesthetized by 2% halothane in O2 and room air (70:30) inhalation by face mask. Holes were placed in the calvarium, dura, and arachnoid mater to expose an area of the superior and lateral cortex 2 mm lateral to the midsagittal suture and 2 mm anterior to the lambda. The CC-PACEs were positioned so as to be jux­ taposed to the pial surface and to occlude the craniotomy, then cemented in place with cyanoacrylic. A thin plati­ num wire (0.2 mm) was placed in the subarachnoid space adjacent to the CC-PACE, 3 mm lateral to the midsagittal suture and 2 mm posterior to the bregma, as a counter­ electrode, and the reference cell was attached cutane­ ously. Current measurements were made as described with a constant applied voltage of 0.05 V relative to SCE. Hypoxemia with hypercarbia was produced by discon­ tinuing the air flow and allowing the animal to rebreathe into the face mask for periods of between 3 and 10 min. To ensure specificity of the signal, 50- 100 U of Cu-Zn superoxide dismutase (SOD; Sigma) in 10 fLl HBSS was injected under the CC-PACE using a Hamilton syringe to test for quenching of the superoxide signal. In addition, some experiments were repeated using PACE material coated with bovine serum albumin instead of cytochrome c. At the termination of some experiments, electrodes were removed and calibrated again to determine the loss of electrode sensitivity.

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ELECTRODE

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Superoxide production with focal ischemia and reperfusion

Adult Sprague-Dawley rats were anesthetized with 2% halothane and placed on a heating blanket to maintain body temperature. The middle cerebral artery occlusion technique used has been described in detail elsewhere (Longa et aI., 1989). This technique has been shown to produce a dense core of ischemia, while the cortical sur­ face monitored in the present study is included in the periinfarct penumbral region (Quast et aI., 1993). Cran­ iotomies were drilled to allow for the placement of the CC-PACE and a platinum wire counterelectrode as de­ scribed. Recordings of current were made as detailed pre­ viously. The animal was inverted and the common ca­ rotid, external carotid, and pterygopalatine arteries li­ gated. A suture was passed up the internal carotid artery to occlude the middle cerebral artery for 1 h. The suture was then withdrawn to allow for reperfusion and moni­ toring continued for an additional hour. Superoxide production with trauma/hypotension/reperfusion injury

Male Sprague-Dawley rats (�450 g) were anesthetized with isoflurane in an anesthetic chamber, intubated, and mechanically ventilated with 1.5-2.0% isoflurane in O2 and room air (30:70) using a volume ventilator (EDCO Scientific). Polyethylene cannulas were placed in both femoral arteries and in one vein for arterial blood pres­ sure recording and controlled hemorrhage and for drug infusion. Rectal temperature was monitored using a tele­ thermometer (Yellow Springs Instruments, Yellow Springs, OH, U.S.A.) and maintained using a thermostat­ ically controlled water blanket (Gaymar, Orchard Park, NY, U.S.A.). A 4-mm craniotomy was trephined 2 mm to the right of the midsagittal suture midway between the lambda and bregma. A plastic adapter for the neuro­ trauma device (see following) was cemented into the cra­ niotomy using cyanoacrylic. Additional craniotomies were drilled on the left of the midline, into which were cemented the CC-PACE and a counterelectrode as de­ scribed. These sites were then covered with dental acrylic. At the completion of surgical preparation, isoflu­ rane concentration was decreased to 1.0-1.5% in O2 and air, and arterial pH, Paco2, and Pao2 were monitored and maintained within normal limits using ventilatory rate and volume adjustments. After the dental acrylic had hard­ ened, the rats were connected to the fluid percussion in­ jury device. The fluid percussion trauma device consists of a Plexiglas cylinder 60 cm long and 4.5 cm in diameter. One end is connected to a hollow metal cylinder housing a pressure transducer (PA856- 100; Statham, Hato Rey, Puerto Rico) and the other end is closed by a Plexiglas piston mounted on O-rings. The transducer housing is connected to the animal via a hollow metal injury tube to the craniotomy. The piston is struck with a 4.8-kg steel pendulum dropped from a preset height. The pressure pulse generated by the device is recorded on a storage oscilloscope that is triggered photoelectrically by a sensor activated by the descent of the pendulum (Dixon et aI., 1987). Rats were subjected to a moderate fluid percussion in­ jury (2.2 atm), and recordings of superoxide production were made. An intravenous injection of a bolus of 8,00024,000 U of Cu-Zn SOD in 1 ml saline was made in some experiments at various times. Mean arterial pressure was

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R. H. FABIAN ET AL.

then lowered to -40 mm Hg by controlled hemorrhage from one of the femoral arterial catheters. After - 15 min of hypotension, the shed blood was reinfused to return arterial pressure to normal.

·1

·2

50 pmol min mm

L

Statistical methods

In all cases, standard deviation was utilized to repre­ sent dispersion of data.

10 min

RESULTS Response of CC-PACE in vitro

The sensitivity of the electrodes used in this study for the experiments described was 58 ± 24 M-mm2 amp - 1 min 1 (n 4). A linear relationship between the superoxide production rate in solution and the maximum current response was evident (data not shown). The loss of electrode sensitivity during experiments as determined by recalibration was 1 1 ± 12% (n 3). The CC-PACE showed no significant current change with increasing concen­ trations of sodium nitroprusside or hydrogen perox­ ide plus increasing concentrations of ferrous sul­ fate. -

-

=

=

Hypoxemia/hypercarhia

A current increase was noted 2 or 3 min following the onset of hypoxemia/hypercarbia (Figs. 1B and 2). A stable baseline current was monitored for 15 min before each experiment. With periods of hyp­ oxemia lasting longer than 5 min, the current did not return immediately to the baseline as it did with shorter periods of hypoxemia, suggesting that su­ peroxide production persisted for up to 15 min. In­ jection of Cu-Zn SOD near or under the electrode reduced the current by up to 83% with an average of 60% (n 3) for a period of a few minutes, while injections of carrier buffer had little effect (Figs. IB and 3). Recordings made with a PACE coated with bovine serum albumin showed a minor increase in current with onset of hypoxemia that was quite small compared with the current seen with the CC­ PACE (Fig. lA). The cytochrome c reduction in­ crease calculated from the current inhibited by SOD was 40.8 ± 12. 0 pmol min - 1 mm - 2 with hypoxemia of 5-min duration. This corresponds to the same reduction rate obtained when the electrode is sub­ merged in a buffer solution in which the rate of production of superoxide per unit volume is 40.0 ± 1 1. 9 nM min-I.

-

-

--

B

A

FIG. 1. Current measurements made during asphyxia with a

platinized carbon electrode (PACE) coated with bovine se­ rum albumin (A) and the cytochrome c-coated PACE (8). The bars indicate the duration of asphyxia. The open arrow indi­ cates the injection of carrier and the filled arrows the injec­ tion of superoxide dismutase solution near the electrode. The reduction rate of cytochrome c relative to the electrode surface area in picomoles per minute per square millimeter is indicated by the vertical bar and the time base by the hori­ zontal bar.

Trauma/hypotension/reperfusion injury

There was an increase in current within a few minutes after fluid percussion injury that reached a plateau after 20 min (n 3). Current increased again 10 min after the the onset of hypotension. There was a third increase in current during reinfu­ sion of shed blood (Fig. 5). Repeated systemic bolus injections of SOD reproducibly reduced the current �

=

�

80 r-------,,-.--�

=

Focal ischemia and reperfusion

In the focal ischemia model, an increase in cur­ rent immediately followed the onset of ischemia and continued until reperfusion (n 1). There was a smaller, secondary increase in the current with reperfusion (Fig. 4). =

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�

60 E E

":c

E

40

o

E 20 0. o

-4

-2

o

2

4 min

6

8

10

F I G. 2. Summary of eight episodes of hypoxemia/ hypercarbia of 5-min duration in three rats. The x-axis rep­ resents the time relative to the onset of asphyxia, which is at o min. The y-axis represents the reduction rate of cy­ tochrome c relative to the electrode surface area in pico­ moles per minute per square millimeter. Error bars represent SOs.

CYTOCHROME c ELECTRODE by as much as 30% for periods of 6-10 min (Fig. 6). In some animals no significant current increase was detected following trauma. In these animals, blood was found to have accumulated around the CC­ PACE.

-1

245

-2

25 pmol min mm

DISCUSSION

The CC-PACE proved to be both sensitive to changes in superoxide levels and responsive to minute-by-minute changes in superoxide levels dur­ ing brain tissue injury in ischemic and traumatic models of brain injury. We confirmed earlier work showing that CC-PACEs responded to superoxide production (xanthine-xanthine oxidase) in vitro (McNeil et aI., 1992). We also demonstrated the CC-PACE responded with increasing current dur­ ing hypoxia and ischemia and that these increases likely represented superoxide production since they were inhibitable with Cu-Zn SOD but not with the vehicle. Incomplete inhibition was found, which could mean either that the delivery of SOD was inadequate or inconsistent or that the electrode is sensitive to free radical species other than superox­ ide. However, the latter is less likely considering the lack of response to nitric oxide or hydroxyl rad­ ical in our in vitro studies and to hydroxyl radical in those of McNeil et ai. (1989). In addition, the fact 1.2 *

1.0

if

30 min

FIG. 4. Current measurements made during focal ischemia and reperfusion. The open arrow indicates the onset of mid­ dle cerebral artery occlusion, and the filled arrow the onset of reperfusion. The reduction rate of cytochrome c relative to the electrode surface area in picomoles per minute per square millimeter is indicated by the vertical bar and the time base by the horizontal bar.

that up to 83% signal inhibition was obtained indi­ cates that the signal was predominantly from super­ oxide reduction of cytochrome c. Systemic bolus injections of SOD were inhibitory to a lesser extent. Native SOD has a very short half-life following sys­ temic bolus injection and does not penetrate the blood-brain barrier in large amounts (Petkau et aI. , 1976), which may have blunted the effect on extra­ vascular superoxide anion concentrations in the brain in the present study. -1

-2

50 pmol min mm

0.8

0.6

�� V�

0.4

0.2

t

15 min

o

2

3

min FIG. 3. Response of oxidation current to injection of 100 U

superoxide dismutase in 10 f.LI of buffer (filled circles) versus injection of carrier alone (open circles) at the electrode. The x-axis represents minutes from the time of injection, which is set at 0 min. The y-axis represents the fraction of oxidation current relative to the current before injection. Error bars represent 8Ds. Asterisks indicate a significant difference by t test (p < 0.05).

A

B

FIG. 5. Current measurements made during and following

fluid percussion brain injury without (A) and with (8) hemor­ rhagic hypotension and reperfusion. The large open arrows indicate the moment of trauma, and the filled arrows indicate the onset of hemorrhagic hypotension and reperfusion, re­ spectively. The small open arrows indicate the systemic in­ jection of superoxide dismutase. The reduction rate of cy­ tochrome c relative to the electrode surface area in pico­ moles per minute per square millimeter is indicated by the vertical bar and the time base by the horizontal bar.

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246 ·1

-2

50 pmol min mm

�

10 min FIG. 6. Current measurements made during and following

fluid percussion brain injury. The large arrow indicates the moment of trauma, and the small arrows indicate the sys­ temic injection of 16,000 U of superoxide dismutase (SOD). A reduction of current is noted following each SOD injection. The reduction rate of cytochrome c relative to the electrode surface area in picomoles per minute per square millimeter is indicated by the vertical bar and the time base by the hori­ zontal bar.

We also demonstrated that traumatic brain injury (TBI) results in superoxide production that contin­ ues for at least 30 min and that hypotension follow­ ing TBI produces further increases in superoxide. Finally, our results indicate that continued hypoten­ sion after TBI is associated with decreasing super­ oxide production and that reinfusion reinitiates su­ peroxide production. Hypotension after TBI is as­ sociated with profound decreases in O availability 2 (De Witt et aI., 1992), which might limit the produc­ tion of oxygen free radicals. The generation of superoxide and other reactive oxygen species in the brain has been studied by others (Kontos and Wei, 1986; Armstead et aI., 1988; Patt et aI., 1988; Nelson et aI., 1992; Globus et aI., 1994). Kontos and Wei (1986) found levels of 3.52 ± 0.72 nmol cm-2 min-lor 35.2 ± 7.2 pmol mm-2 min- 1 following fluid percussion injury in the cat. In these studies, superoxide production was measured as the SOD-inhibitable fraction of nitro­ blue tetrazolium reduction, which was capable of measurements at only a few time points. These val­ ues are within the same general order of magnitude that we calculate for the rate of cytochrome c re­ duction at the electrode. The time course of pro­ duction determined by other investigators is also similar to our findings. There is evidence that superoxide is involved in tissue injury. Other oxygen radicals are probably involved, but superoxide is damaging in itself and contributes to the formation of other toxic radicals. Antioxidants such as SOD have proven to be pro­ tective in experimental models of tissue injury, and this has led to clinical trials using SOD coupled to polyethylene glycol for the treatment of brain

J Cereb Blood Flow Metab, Vol. 15, No. 2, 1995

trauma (Muizelaar et aI., 1993). However, much is not known about superoxide generation and its bi­ ological effects because of the difficulties involved in its measurement, especially in vivo. The application of the CC-PACE to in vivo work should provide considerable additional information concerning the time course and other aspects of su­ peroxide production in the brain and other tissues. The role of superoxide anion in injury to the brain and its vasculature that occurs in ischemia and trauma will be more readily addressed with a method for measuring relative levels of superoxide on an ongoing basis. Acknowledgment: Dr. Calum McNeil provided the nec­ essary PACE material and useful advice. We thank Neng Huang, H. Charmaine Rea, and Kimberly Miller for ex­ cellent technical assistance and Dr. Donald Prough for useful advice. This work was funded by a grant from the Amyotrophic Lateral Sclerosis Association and by NIH grant NS 19355.

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