Direct Assessments of the Antioxidant Effects of Propofol Medium Chain Triglyceride/Long Chain Triglyceride on the Brain of Stroke-prone Spontaneously Hypertensive Rats Using Electron Spin Resonance Spectroscopy

Propofol MCT/LCT (1% propofol injection, Maruishi) was purchased from Maruishi Pharmaceutical (Osaka, Japan). MCT/LCT was prepared with soybean oil, MCT, lecithin from chicken eggs, glycerol, and sodium oleate. Propofol LCT (1% Diprivan injection) was purchased from AstraZeneca Ltd. (Osaka, Japan). Xanthine oxidase (XO) (grade III: from buttermilk, chromatographically purified suspension in 2.3 m (NH⁴)²SO⁴, 10 mm sodium phosphate buffer [pH 7.8], containing 1 mm EDTA and 1 mm sodium salicylate), xanthine, and superoxide dismutase were obtained from Sigma (St. Louis, MO). Hydrogen peroxide (H²O²) and FeSO⁴were obtained from Wako Chemical (Osaka, Japan). 5,5-Dimethyl-1-pyrroline-N -oxide (DMPO) was purchased from Labotec (Tokyo, Japan). MC-PROXYL was synthesized from 3-carboxy-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (carboxy-PROXYL; Tokyo Kasei, Tokyo, Japan) and diazomethane (Wako Chemical) was synthesized by a method described previously.20–22 

HO^·were generated by the Fenton reaction (H²O²plus FeSO⁴) as described previously.30,31HO^·were generated by ultraviolet irradiation of H²O²as described previously.32,33O^2·−were generated by the xanthine–XO system by methods also described previously.18All solutions were prepared in 0.1 m phosphate-buffered saline (pH 7.2). ESR spin trapping was conducted with a ROS generating system containing DMPO. ESR observations were performed with a JES-RE 3X, X-band spectrometer (JEOL, Tokyo, Japan) connected to a WIN-RAD ESR Data Analyzer (Radical Research, Tokyo, Japan) at the following instrument settings: microwave power, 8.00 mW; magnetic field, 335.638 ± 5 mT; field modulation width, 0.079 mT; receiver gain, 400; sweep time, 1 min; and time constant, 0.03 s. Hyperfine coupling constants were calculated based on resonance frequency, measured with a microwave frequency counter, and resonance field, measured with a JEOL ES-FC5 field measurement unit. To quantify the spin adducts detected, we obtained ESR spectra for manganese oxide standards. After the ESR spectra were recorded, the signal intensity, expressed as relative height, was normalized against the signal intensity of the manganese oxide standard.30,31All experiments were repeated a minimum of four times.

The procedures used in this study were in accordance with the guidelines of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals  (publication 85-23, revised 1985), and our protocols were approved by Animal Care Committees of Kanagawa Dental College (Yokosuka, Japan). Male Wistar-Kyoto rats (WKYs) and SHRSPs, weighing approximately 100–120 g, were purchased from Japan SLC (Hamamatsu, Japan).

In preliminary experiments, we confirmed that WKYs and SHRSPs were successfully anesthetized with a minimal concentration of pentobarbital (30 mg/kg, intravenous) for in vivo  L-band ESR. Therefore, WKYs and SHRSPs were anesthetized with pentobarbital (30 mg/kg, intravenous) or propofol MCT/LCT (20 mg/kg, intravenous) and used for in vivo  L-band ESR. We then analyzed the head region of WKYs or SHRSPs with in vivo  L-band ESR analysis as described previously.21,22The ESR spectra from SHRSPs were obtained with a L-band ESR spectrometer (JES-RE-3 l; JEOL, Akishima, Japan) equipped with a four-window loop-gap resonator, under the following conditions: microwave power, 10 mW; magnetic field, 50.0 ± 10 mT; field modulation width, 1 mT; receiver gain, 79–320; sweep time, 0.5 min; and time constant, 0.03 s. All experiments were repeated a minimum of five times.

The decay rate of MC-PROXYL was used, under the same conditions as the L-band ESR assay, to ascertain whether propofol MCT/LCT treatment had any effect on cerebral blood flow (CBF) in the brain of SHRSPs and WKYs. CBF was measured by a laser Doppler flowmeter (TBF-LN1; Unique Medical, Tokyo, Japan) using a laser Doppler flowmetry probe (LP-C; Unique Medical). It has previously been reported that urethane anesthetic has a wide safety margin and has little influence on normal blood pressure and the general circulation.34,35As a consequence, rats were anesthetized with urethane (1.2 g/kg, intraperitoneal) before CBF measurements. Each rat was fixed on a wooden board (18 × 25 cm) in the prone position. All limbs were fixed at an angle of 45° to the body midline with adhesive tape. The head was also fixed to the board with adhesive tape. A laser Doppler flowmetry probe was glued directly onto the surface of the skull. The output signals from the flowmeter were recorded on a computer hard disk through an analog–digital converter and displayed simultaneously on the monitor. CBF was recorded and analyzed using data analysis software (Chart version 4.2; AD Instruments, Inc., Colorado Springs, CO). After CBF had stabilized at a baseline, propofol MCT/LCT (20 mg/kg) was administered by tail vein injection. CBF was then continuously monitored over a 10-min period, the same time course as L-band ESR studies. Pericranial temperature, arterial oxygen tension, arterial carbon dioxide tension, and blood pressure were controlled and remained at consistent levels across all groups before or during CBF measurements. All of the traces presented are typical results. All experiments were repeated a minimum of four times.

Statistical analyses were performed using Statcel (OMS, Saitama, Japan). Analyses demonstrated that data arising from this experiment were normally distributed. Results are presented as mean ± SD. Two-way analysis of variance was used to compare the averages of six concentration levels and between agents. The Bonferroni/Dunn test was used for multiple comparisons. Regression analysis was applied to data obtained from the L-band ESR analysis. The Student t  test was used to compare averages between WKY and SHRSP. A P  value of less than 0.05 was considered to be statistically significant.

In the current study, we investigated the effects of propofol MCT/LCT on HO^·, which had been generated from the Fenton reaction, by ESR spin trapping with DMPO.

As reported previously,30,31,36after the addition of H²O²to FeSO⁴, we primarily observed a characteristic DMPO-OH spin adduct spectrum with hyperfine splitting giving rise to four resolved peaks (figs. 1A and B, control). With propofol MCT/LCT (56.09–5,609 μm) pretreatment of FeSO⁴and subsequent addition of H²O², it was apparent that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 1A). However, with pretreatment using the equivalent vehicle (MCT/LCT), it was also demonstrated that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 1B). These data indicate that, within this system, propofol MCT/LCT markedly inhibits HO^·generation from the Fenton reaction as a result of scavenging, both by the propofol MCT/LCT and by the vehicle. It was also clear that both propofol MCT/LCT and the vehicle reduced HO^·generation in a dose-dependent manner (fig. 1C).

In the next study, we investigated the effects of propofol MCT/LCT on HO^·, which had been generated from ultraviolet irradiation of H²O², and by ESR spin trapping with DMPO.

As reported previously,32,33,36after ultraviolet irradiation of H²O², we primarily observed a characteristic DMPO-OH spin adduct spectrum with hyperfine splitting giving rise to four resolved peaks (figs. 2A and B, control). When H²O²was pretreated with propofol MCT/LCT (56.09–5,609 μm) followed by ultraviolet irradiation, it was apparent that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 2A). When the experiment was repeated using the equivalent vehicle pretreatment, it was evident that the DMPO-OH signal was not significantly different at low concentration (figs. 2B and C) but was reduced at high concentrations (>560.09 μm) of vehicle (figs. 2B and C). Interestingly, the inhibitory effects of propofol MCT/LCT (>224.36 μm) were significant stronger than the vehicle alone (fig. 2C). These data suggest that propofol MCT/LCT directly inhibited HO^·generation in a manner that was independent of the scavenging effects of the vehicle (fig. 2C).

In the current study, we investigated the effects of other propofol formulations, such as propofol LCT (Diprivan) on HO^·generated from the Fenton reaction by ESR spin trapping with DMPO.

When FeSO⁴was pretreated with propofol MCT/LCT (56.09–5,609 μm) and the subsequent addition of H²O², it was apparent that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 3A), as shown in figure 1. With equivalent propofol LCT (56.09–5,609 μm) pretreatment, it was also shown that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 3B). We also observed that the inhibitory effects of a high concentration of propofol MCT/LCT (>560.9 μm) were significantly stronger than propofol LCT (fig. 3C). Collectively, these data strongly indicate that, within this system, the vehicle can account for the scavenging effects of HO^·on the concentration of propofol MCT/LCT (fig. 1C).

In the next study, we also investigated the effects of propofol LCT on HO^·generated from ultraviolet irradiation of H²O²and ESR spin trapping with DMPO.

As shown in figure 2, with propofol MCT/LCT (56.09–5,609 μm) pretreatment of H²O²and subsequent ultraviolet irradiation, it was apparent that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 4A). With equivalent propofol LCT pretreatment, it was also shown that the DMPO-OH signal was reduced in a dose-dependent manner (fig. 4B). The inhibitory effects of propofol MCT/LCT were no different in propofol LCT (fig. 4C). These data suggest that propofol LCT can also inhibit HO^·generation directly (fig. 4C), in a manner similar to that propofol MCT/LCT (fig. 2C).

We also investigated the effects of propofol MCT/LCT on XO-mediated O^2·−generation, as measured by ESR spin trapping with DMPO. As reported previously,36after addition of xanthine to XO, we primarily observed a characteristic DMPO-OOH adduct spectrum with hyperfine splitting giving rise to 12 resolved peaks (fig. 5A, a). These signals were quenched by 150 U/ml superoxide dismutase, thus confirming that they were derived from O^2·−(fig. 5A, b). With propofol MCT/LCT (original liquid; 5,609 μm) pretreatment of XO and subsequent addition of xanthine, however, we did not observe any alterations in the ESR spectrum, which exhibited identical DMPO-OOH spin adducts (fig. 5A, c). Data showed that propofol MCT/LCT had no effect on O^2·−generation from xanthine–XO in this system (fig. 5B).

The effects of propofol MCT/LCT on SHRSP-induced oxidative stress in the brain were investigated using an in vivo  L-band system to monitor the decay rate of MC-PROXYL as a nitroxyl spin probe. The decay rate constants of MC-PROXYL were significantly greater in the head region of SHRSPs than in WKYs anesthetized with pentobarbital (30 mg/kg, intravenous) (fig. 6A). Furthermore, the decay rate constant of SHRSPs was significantly faster than in WKYs, suggesting that oxidative stress in the brain of SHRSPs was higher compared with that in WKYs, the latter being the oxidative stress model reported in our previous study.21,22However, we did not observe any alterations in the decay rate constant of MC-PROXYL in the head region of either SHRSPs or WKYs anesthetized with propofol MCT/LCT (20 mg/kg, intravenous) (fig. 6B). These results indicated that the induced oxidative stress in the SHRSP brain was reduced by propofol MCT/LCT but not by pentobarbital.

Figure 7shows the typical effects of propofol MCT/LCT on CBF in WKYs (A) and SHRSPs (B). Upon propofol MCT/LCT administration, we observed a rapid decline in CBF, both in WKYs and in SHRSPs; CBF levels reached the same minimum level in the two models within a few seconds (figs. 7A and B). Furthermore, it was observed that there was no significant difference between the WKYs and SHRSPs when CBF levels were compared 10 min after propofol MCT/LCT injection (figs. 7A and B), equivalent to the period of time monitored in the L-band ESR study (fig. 6), although CBF levels in WKYs were higher than in SHRSPs immediately before the injection of propofol MCT/LCT (fig. 7C). These results indicated that anesthesia undertaken with propofol MCT/LCT during L-band ESR measurements had no significant effect on the levels of CBF in either WKYs or SHRSPs.

Propofol anesthesia has been associated with lower intracranial pressure and cerebral swelling than volatile anesthesia in brain tumor patients undergoing craniotomy.37,38The potential neuroprotective effect of propofol may be mediated by its antioxidant properties, which have been shown to play a role in apoptosis, ischemia–reperfusion injury, and inflammatory-induced neuronal damage.39,40However, questions and controversy remain regarding whether propofol modulates ROS generation and ROS-induced oxidative stress in the brain. Many existing studies have assessed the antioxidant effects of propofol by measuring lipid peroxidation.7–9However, these previous studies do not demonstrate the direct effects of ROS generation. As a consequence, we have performed studies to assess the direct effects of ROS generation, such as HO^·and O^2·−, using ESR. Our studies provide direct evidence that propofol MCT/LCT inhibits HO^·generation but not O^2·−generation from xanthine–XO (figs. 1–5).

First, we can confirm that propofol MCT/LCT inhibited the generation of HO^·from the Fenton reaction in a dose-dependent manner (figs. 1A and C). Surprisingly, the equivalent vehicle solution also reduced HO^·generation in a dose-dependent manner (figs. 1B and C) in this system. It is well established that HO^·can be generated from a reaction known as the biologic Fenton reaction.

The existence of this type of reaction in disease states was indicated by the ability of an iron chelator such as Desferal to reduce the high-intensity signal of the DMPO-OH spin adduct, thus suggesting the generation of HO^·.41,42The generation of HO^·from the Fenton reaction has been shown to be a critical factor in various ROS-induced brain diseases.43Our current results provide the first direct evidence of the antioxidant properties of a vehicle such as MCT/LCT. The effects of the vehicle are most probably due to scavenging HO^·from the biologic Fenton reaction in tissues such as the brain. Further studies are urgently required to study how MCT/LCT scavenges HO^·generated via  the Fenton reaction or ultraviolet irradiation of H²O²in vitro . It would also be important to investigate how MCT/LCT scavenges HO^·from the brain of SHRSPs in vivo .

Second, we investigated the effect of propofol MCT/LCT on other HO^·generating systems using ultraviolet irradiation of H²O², a well-known Fe²⁺-independent reaction.32,33Interestingly, propofol MCT/LCT inhibited the generation of HO^·via  ultraviolet irradiation of H²O²in a dose-dependent manner (figs. 2A and C). An equivalent vehicle also reduced the generation of HO^·in a dose-dependent manner (figs. 2B and C) in this system, but the inhibitory effects observed were smaller than with propofol MCT/LCT (fig. 2C). These results suggested that the scavenging effects of propofol MCT/LCT on HO^·in this system would also be dependent on the effects of the vehicle. However, the high concentration of propofol MCT/LCT (> 224.36 μm) could be directly inhibited the HO^·generated by ultraviolet irradiation of H²O².

Our next study was performed to compare the antioxidant characteristics of propofol MCT/LCT with other types of propofol in an LCT emulsion, propofol LCT (Diprivan), by measuring the HO^·generating system by use of the ESR technique. Propofol MCT/LCT inhibited the generation of HO^·via  the Fenton system in a dose-dependent manner (figs. 3A and C). With the equivalent propofol LCT, we also observed a reduction in HO^·generation in a dose-dependent manner (figs. 3B and C), although the inhibitory effects were smaller than with propofol MCT/LCT (fig. 3C). The powerful effects of propofol MCT/LCT on the generation of HO^·via  the Fenton system are likely to be caused by scavenging of the vehicle (fig. 1C). It is likely that a vehicle such as propofol LCT exerts smaller antioxidant effects compared with propofol MCT/LCT. In addition, propofol LCT also directly inhibited the generation of HO^·via  ultraviolet irradiation of H²O²system (fig. 4C) in a similar manner to propofol MCT/LCT (figs. 2 and 4). From these results, it is possible that the dual antioxidant properties of propofol MCT/LCT would be stronger than those of propofol LCT alone, especially in terms of the biologic Fenton system. This is likely due to the scavenging effects of vehicle, such as MCT/LCT (fig. 1C).

Excellent and predictable recovery conditions, as well as minimal postoperative side effects, make propofol particularly suitable in awake craniotomies.37,38However, little is known about the potential effects of propofol on oxidative stress in the brain of in vivo  experimental models. In vivo  L-band ESR spectroscopy is a highly useful technique for the noninvasive investigation of redox status in living organisms. Our laboratories have previously reported that quantitative ESR analysis using MC-PROXYL has the potential to be highly useful for understanding redox status under conditions of oxidative stress in the rodent brain.20–22Severe hypertension and cerebrovascular diseases develop in SHRSPs.44ROS generation occurs after reperfusion, and this ROS-induced oxidative stress can greatly damage neurons in SHRSPs.45We evaluated high oxidative stress in the brain of SHRSPs using in vivo  L-band ESR.21,22Consequently, it is clear from our SHRSP investigations that ESR techniques are highly useful in the assessment of the antioxidant properties of propofol MCT/LCT for use clinically. Indeed, we confirmed that oxidative stress in the brain of SHRSPs was higher compared with that in WKYs anesthetized with pentobarbital (fig. 6A). When propofol MCT/LCT was used as an anesthetic, rather than pentobarbital, the level of oxidative stress in SHRSPs became similar to that seen in WKYs (fig. 6B), suggesting that propofol MCT/LCT reduced SHRSP-induced oxidative stress in the brain. Consequently, propofol MCT/LCT could be particularly useful in adjusting levels of anesthesia in cases of ROS-induced brain disease.

Nitroxyl spin probes are usually eliminated by a combination of excretion via  the kidney and bioreduction to the hydroxyl amine. The rate of elimination by the kidney seems to follow the usual pharmacologic pattern for the excretion of drugs, with those that tend to stay in the vascular system being excreted rapidly.46By contrast, neutral water-soluble nitroxyl spin probes, exhibiting an intermediate rate of urinary excretion, and lipophilic nitroxyl spin probes, such as MC-PROXYL used in this study (fig. 6), exhibit much reduced excretion via  the kidney.46,47We have already confirmed that the metabolism of MC-PROXYL in the brain can be divided into phase I (vascular system) and phase II (reduced excretion via  kidney), according to a two-compartment model of distribution using in vivo  L-band ESR analysis.21,22Although several possible mechanisms should be considered to be responsible for the increased signal decay, a number of studies of L-bands in living animals have been associated with redox state, metabolism, and biologically relevant free radical generation.48–53Recently, it was shown that oxygen concentration, antioxidant content, and free radicals alter the signal decay rate of nitroxyl radical.46,49In the current study, we first measured the decay rate constant of the phase I component, which was dependent on bioreduction by the vascular system, of MC-PROXYL metabolism in the brain using the L-band ESR technique (fig. 6). Metabolism was probably in phase I because of the presence of ascorbate, which is a strong reducing agent of nitroxyl spin probes in the vascular system of the brain.46Therefore, we investigated the effects of propofol MCT/LCT on CBF, which is known to influence the bioreduction of MC-PROXYL in the blood circulation of the brain, in WKYs and SHRSPs. CBF in both WKYs and SHRSPs was immediately reduced by propofol MCT/LCT treatment. There were no significant differences in CBF level when compared between WKYs and SHPSPs 10 min after the injection of propofol MCT/LCT, a time period equivalent to the L-band ESR study (fig. 7). Therefore, results suggested that CBF is not likely to affect the bioreduction of MC-PROXYL in the brain of either WKYs or SHRSPs. The decay rate constant of MC-PROXYL was recovered when animals were anesthetized with propofol MCT/LCT but not pentobarbital (fig. 6). Collectively, this suggests that propofol MCT/LCT could reduce high oxidative stress in the brain of the SHRSP, a valued pathologic model of ischemic brain injury such as stroke.

If we turn our attention to how our results may relate to the clinic, it is important to consider the relative dose levels of the treatments used in the current study. The smallest concentration of propofol used was 10–30 μm, compared with the minimum concentration of propofol MCT/LCT of 56.09 μm used in our in vitro  experiments (figs. 1–4). However, the scavenging effects of low concentrations of propofol MCT/LCT (< 112.18 μm) were observed to be dependent on the effects of vehicle (fig. 1C), in a manner similar to that of propofol LCT (fig. 3). Brain tissue harvested from SHRSPs is known to exhibit high levels of oxidative stress.21,22,28,29Our current study has shown that propofol MCT/LCT treatment resulted in the recovery of normal levels of oxidative stress in the brain of SHRSPs (figs. 6 and 7). These results suggest the possibility that propofol MCT/LCT may exhibit useful antioxidant activity for clinical anesthesia for surgical aspects of brain disease such as craniotomy. However, further studies will be required to examine the clinical importance of propofol MCT/LCT, especially the MCT/LCT vehicle, on anesthesia in cases of ROS-induced brain disease.

In conclusion, our studies have demonstrated that propofol MCT/LCT and vehicle exhibits antioxidant properties by scavenging HO^·and can reduce high oxidative stress in the brain of SHRSPs. This suggests that propofol MCT/LCT can prevent ROS-related injuries in the brain, such as stroke, and thereby provide significant advantage for neuroprotection during anesthesia for craniotomy in patients with brain disease.

The authors thank Hirohisa Arakawa, D.D.S., Ph.D. (Professor, Department of Health Science Division of Oral Health, Kanagawa Dental College, Yokosuka, Kanagawa, Japan), for statistical advice.