JOURNAL HOME CME HOME THIS MONTH PAST ISSUES ETOC COLLECTIONS
AUTHORS REVIEWERS EDITORIAL BOARD FEEDBACK RSS HELP
A&A International Anesthesia Research Society
QUICK SEARCH:   [advanced]


     

Anesth Analg 2008; 106:1242-1249
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e318164f2a5
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, B.
Right arrow Articles by Li, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, B.
Right arrow Articles by Li, W.
Related Collections
Right arrow Mechanisms
Right arrow Neuroanesthesia
Right arrow Preclinical Pharmacology
Right arrow Pharmacology

SURGICAL ANESTHESIOLOGY

Section Editor:
Adrian W. Gelb

Desflurane Affords Greater Protection Than Halothane in the Function of Mitochondria Against Forebrain Ischemia Reperfusion Injury in Rats

Bing Zhang, MD, PhD, Xia Wei, MD, Xiaoguang Cui, MD, PhD, Huacheng Zhou, MD, PhD, Wengang Ding, MD, PhD, and Wenzhi Li, MD, PhD

From the Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, China.

Address correspondence and reprint requests to Dr. Wenzhi Li, MD, PhD, Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, 150086 Harbin, China. Address e-mail to Wenzhili9{at}126.com.

Abstract

BACKGROUND: Halothane and desflurane have been shown to attenuate neuronal injury; however, the effects of these anesthetics on mitochondria are unclear. We investigated whether halothane and desflurane affect the function of mitochondria after cerebral ischemia in rats.

METHODS: Forty male Wistar rats were randomly divided into four groups (n = 10 each): sham group; 1.5 minimal alveolar concentration (MAC) halothane group; 1.0 MAC desflurane; and 1.5 MAC desflurane group. Forebrain ischemia was induced after 40-min inhalation of 1.5 MAC halothane, 1.0 MAC or 1.5 MAC desflurane by clamping the bilateral common carotid arteries and decreasing arterial blood pressure. After isolation of the brain mitochondria, mitochondrial membrane permeability was assayed spectrophotometrically with 40–200 µM Ca2+, and mitochondrial membrane potentials were measured by a fluorospectrophotometer with the addition of rhodamine 123. The activities of mitochondrial respiratory chain complexes were also assayed spectrophotometrically.

RESULTS: The results showed obvious mitochondrial swelling, loss of membrane potential with the addition of Ca2+, and inhibition of the activities of complexes I + III and IV after forebrain ischemia reperfusion injury. Compared with the 1.5 MAC halothane group, 1.0 and 1.5 MAC desflurane reduced mitochondrial swelling by 23.9% (P < 0.001) and 23.2% (P < 0.001), whereas membrane potential dissipation was suppressed by 22.4% (P = 0.013) and 20.4% (P = 0.027). The activities of complexes I + III and IV were better preserved in 1.0 MAC and 1.5 MAC desflurane groups than in the 1.5 MAC halothane group by 34.6% (P = 0.027), 38.7% (P = 0.011), 53.9% (P = 0.009), and 55.8% (P = 0.007), respectively.

CONCLUSIONS: Desflurane shows better preservation of mitochondrial function at 4 h after cerebral ischemia reperfusion injury, indicated by inhibition of mitochondrial swelling, increase of membrane potential, and improvement of functions of mitochondria respiratory complexes I + III and IV when compared with halothane.

There is increasing evidence that mitochondria plays an important role in mediating either necrotic or apoptotic neuronal cell death during ischemia reperfusion injury.1 A number of mitochondrial changes, including disruption of mitochondrial membrane permeability and alterations of the mitochondrial membrane potential (MMP), have been observed that lead to release of intramitochondrial proteins, in which cytochrome c is the best characterized. When present in cytosol, cytochrome c can combine with Apaf 1 and caspase 9 to form the apoptosome, which activates other caspases that finally dismantle the cell.2,3 In addition, during ischemia reperfusion injury, mitochondria can produce reactive oxygen species,4 which may cause nonspecific damage to lipids, proteins, DNA, and other important components of cells, including many respiratory enzymes, leading to a decline in the efficiency of oxidative phosphorylation, dysfunction of proton transfer, and electro leak from the respiratory chain, in turn, aggravating the production of free radicals and hence potentially causing cell death.

Volatile anesthetics, such as halothane, isoflurane, and desflurane, are commonly used in neuroanesthesia, and have demonstrated neuroprotective properties in some experimental models of cerebral ischemia.5–7 Desflurane is a new inhaled anesthetic with rapid induction and fast recovery due to its low blood/gas solubility ratio.8 Some studies have shown that desflurane improves neurological outcome after transient incompleted cerebral ischemia in rats9 and increases tissue oxygenation during transient middle cerebral artery occlusion in humans.10 However, its mechanism of action is still unclear, and few studies have reported the effects of desflurane on mitochondria following cerebral ischemia. The purpose of the present study was to determine whether desflurane could influence the functions of mitochondria at 4 h after cerebral ischemia reperfusion injury and to compare such potential effects with that afforded by halothane.

METHODS

Animals and Preparation
All experimental procedures were approved by the Animal Care and Use Committee at the Harbin Medical University and were performed in compliance with the University Institutes of Health guidelines on the ethical use of animals. All measures were taken to reduce animal suffering and numbers of animals used in this study.

Male Wistar rats (250–300 g) were fasted but allowed access to water overnight. All animals were anesthetized with 4% halothane and tracheally intubated. Their lungs were then mechanically ventilated with 1%–1.5% halothane in 30% O2. The right femoral artery and vein were cannulated for arterial blood pressure (BP) measurements, arterial sampling of blood gases and blood withdrawal. Bilateral common carotid arteries were isolated from the carotid sheathes via a ventral midline incision and gently dissected free of surrounding nerve fibers. Before the beginning of forebrain ischemia, baseline measurements of BP, heart rate, temperature, arterial blood gas values, and blood glucose concentration were performed. Tidal volume was adjusted to maintain blood gases in the normal range (pH 7.35–7.45, Paco2 35–45 mm Hg, Pao2 >90 mm Hg). Vecuronium was given as a continuous infusion (0.1 mg · kg–1 · min–1) to maintain neuromuscular blockade. Thermistor probes were inserted into the temporal muscle11 and rectum to monitor the pericranial and rectal temperatures, respectively. The pericranial and rectal temperatures were maintained at 37.5°C ± 0.2°C by a heating pad and a small warming lamp placed above the animal’s head and body. An electroencephalogram was recorded continuously using subdermal needle-type electrodes placed over both hemispheres at the parietotemporal versus frontal cortex recording sites with a Bio-amplifier. All physiologic data were monitored and recorded using Chart 5 software through the PowerLab/16SP system (ADInstruments, Australia).

Forebrain Ischemia
At the end of the preparation, the animals were randomly assigned to one of the following groups: 1.5 minimal alveolar concentration (MAC) halothane (n = 10): anesthesia maintained with 1.1% halothane in O2 (Fio2 = 0.3); 1.0 MAC desflurane (n = 10): animals received 5.7% desflurane and O2 (Fio2 = 0.3); 1.5 MAC desflurane (n = 10): animals received 8.6% desflurane and O2 (Fio2 = 0.3); sham-operated (n = 10): animals received 1.1% halothane in O2 (Fio2 = 0.3) and sham-treatment, i.e., no ischemia and clamping. To induce forebrain ischemia, the mean arterial BP (MAP) was decreased by withdrawing blood from the femoral vein into a heparinized syringe and held at 37.5°C. When MAP had decreased to 45 mm Hg, both common carotid arteries were clamped for 10 min by microaneurysm clips.12 Forebrain ischemia was confirmed by the isoelectric electroencephalogram. During the ischemic period, MAP was maintained at 45 ± 2 mm Hg by withdrawal of blood or reinfusion of the withdrawn blood through the venous catheter. At the end of the 10-min ischemic period, both microaneurysm clips were removed and MAP was normalized by reinfusion of the withdrawn blood. Reperfusion in each artery was verified visually. Blood gas analysis was performed 15 min after the end of the reperfusion period. Before discontinuation of anesthesia, the vascular catheters were removed, and the wounds were sutured. All wounds were infiltrated with 0.25% bupivacaine (total dose 0.5 mg). The endotracheal tube was removed when the rat showed a withdrawal response to endotracheal suction and adequate spontaneous breathing. The animals were then kept in a warm, humidified chamber for about 30 min and then transferred to the animal care facility.

Isolation of Brain Mitochondria
Rat brain mitochondria were isolated as described by Kristian et al.13 with minor modifications. Rats were decapitated 4 h after reperfusion/sham operation, the brain was rapidly removed, and the cerebellum and underlying structures were removed, and the remaining brain tissue was used. Forebrain was minced and homogenized in 6 mL of isolation buffer containing 225 mM mannitol, 75 mM sucrose, 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), and 1 mM K2EDTA (pH 7.4, at 4°C). The resulting homogenate was centrifuged at 1330g for 3 min. The supernatant was decanted, and the pellet was resuspended in half of the original volume and recentrifuged as above. The pooled supernatant was centrifuged at 17,000g for 10 min. The supernatant from this centrifugation step was decanted, and the pellet obtained was resuspended in 15% Percoll (Pharmacia, USA) and layered into centrifuge tubes containing a preformed two-step discontinuous density gradient consisting of 23% Percoll on top of 40% Percoll. The gradients were centrifuged at 37,000g for 20 min (HITACHI 55P-7, Japan). The mitochondrial fraction, located at the interface between the two bottom layers, was carefully removed and diluted 1:5 in isolation buffer and centrifuged at 17,000g for 10 min. The supernatant was decanted and the pellet resuspended in 3 mL isolation buffer. Aliquots were removed for protein determination and bovine serum albumin was added to the mitochondria (10 mg/mL, 0.5 mL/forebrain) to bind any free fatty acids, which can uncouple brain mitochondria.14 After the final centrifugation at 6900g for 10 min, the resulting mitochondrial pellet was then resuspended in 1 mL isolation medium without EDTA. All homogenization and centrifugation steps were performed at 4°C. The content of mitochondria was determined as the concentration of proteins measured by the Bradford15 method with bovine serum albumin as standard.

Electron Microscopy
The method of brain mitochondria preparations used in this experiment was assessed by electron microscopy. An aliquot of mitochondrial suspension in the sham group was removed and centrifuged at 10,000g for 5 min. The mitochondrial pellet was fixed with 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, at 4°C), and postfixed with 1% osmium tetroxide. The samples were dehydrated through increasing concentrations of ethanol, and embedded in Epon 812. Ultrathin sections were cut at 80 nm with an LKB microtome (Bromma, Sweden), stained with uranyl acetate-lead citrate and viewed with a JEM1200 transmission electron microscope (JEOL, Japan).

Permeability Transition Pore Activity
Permeability transition pore (PTP) opening was assayed spectrophotometrically as previously described.16,17 The mitochondria were used immediately after isolation to measure their swelling. Brain mitochondria (0.5 mg protein/mL) were prepared in the assay buffer containing 125 mM sucrose, 50 mM KCl, 2 mM KH2PO4, and 10 mM HEPES. Swelling was initiated by the addition of 40–200 µM CaCl2 to the reaction system. The swelling was evaluated by the spectrophotometric method at 540 nm (30°C) with spectrophotometers (UNICO 2100UV, China). Changes of absorbance in 5 min indicated mitochondrial swelling because of PTP opening. Cyclosporin A at 5 µM was used to test whether permeability pore opening was occurring.

Determination of Mitochondrial Membrane Potential
MMP was evaluated as per Emaus et al.18 from the uptake of the fluorescent cationic dye rhodamine 123 (Rh123), which accumulates electrophoretically into energized mitochondria in response to their negative inside membrane potential. The isolated mitochondria (0.5 mg protein/mL) were incubated in the assay buffer containing 225 mM mannitol, 70 mM sucrose, and 5 mM HEPES (pH 7.2). The discharge of Rh123 was induced by 40 µM CaCl2. MMP was assessed by a fluorospectrophotometer (Shimadzu RF-510, Japan) with excitation at 503 nm and emission at 525 nm after addition of 0.3 µM Rh123 in 5 min at 25°C.

Activity of Mitochondrial Respiratory Enzymes
The measurement of the specific activity of the complexes of the respiratory chain was performed spectrophotometrically, as described by Chuang et al.19 and Brusque et al.20 A total of 100, 200, and 50 µg of mitochondrial protein was used to determine the activities of complexes I + III, II + III, and IV respectively. Assays of all complexes were performed at 37°C. The volume of reaction was 1.0 mL in the case of each complex.

Nicotinamide Adenine Dinucleotide Cytochrome c Reductase (Complexes I + III)
The activity was determined by the reduction of oxidized cytochrome c measured at 550 nm and was calculated as the difference in the presence or absence of rotenone. The activity was assayed in 50 mM K2HPO4 buffer (pH 7.4) containing 1.5 mM KCN, 1.0 mM β-nicotinamide adenine dinucleotide, and 100 µg of protein of mitochondrial suspension in the presence or absence of rotenone (20 µM). The reaction was initiated by adding 50 µM cytochrome c, and absorbance at 550 nm was measured over the first 3 min at 37°C. The molar extinction coefficient of cytochrome c at 550 nm is 19.1 mM–1 · cm–1.

Succinate Cytochrome c Reductase (Complexes II + III)
The activity was performed in 40 mM K2HPO4 buffer (pH 7.4) containing 20 mM succinate, 1.5 mM KCN, and 200 µg of protein of mitochondrial suspension. After 5 min of incubation at 37°C, the reaction was initiated by adding 50 µM cytochrome c, and absorbance at 550 nm was measured over the first 3 min at 37°C.

Cytochrome c Oxidase (Complex IV)
The activity was estimated by recording the oxidation of reduced cytochrome c at 550 nm. Complex IV activity was measured by following the decrease in absorbance due to the oxidation of the previously reduced cytochrome c at 550 nm ({epsilon} = 19.1 mM–1 · cm–1). The reaction buffer contained 10 mM K2HPO4 buffer (pH 7.4), 0.6 mM n-dodecyl-β-d-maltoside, and 50 µg mitochondrial protein. The initial rate of cytochrome c reduction was used for the calculation of the activity. Enzymatic activities of mitochondrial respiratory chain complexes were expressed as nanomoles per minute per milligram of protein. All reagents used in enzyme assays were purchased from Sigma.

Statistical Analysis
Parametric data, i.e., physiologic variables, activity of respiratory enzymes were shown as mean ± sd. For these data, comparisons were performed using one-way analysis of variance with Tukey correction for post hoc comparisons among multiple experimental groups. P < 0.05 was considered to be statistically significant.

RESULTS

Physiologic Variables During Ischemia–Reperfusion
Table 1 summarizes the physiologic variables in each animal group. MAP was held at 45 ± 2 mm Hg during ischemia. There were no significant differences in other physiologic variables throughout the experiment in all groups.


View this table:
[in this window]
[in a new window]

  		Table 1. Physiological Variables (Mean ± sd)

 

Electron Microscopy
The ultrastructure of mitochondria isolated by the above method is shown in Figure 1. The electron micrographs indicated that mitochondria had very well preserved morphological integrity. The mitochondria were elongated (Fig. 1A) or round (Fig. 1B) and had numerous transverse cristae, which exhibited parallel alignment. The outer and inner mitochondrial membranes were clearly distinguishable. The matrixes were uniformly electron dense with no apparent damage to the inner structure.


Figure 132
View larger version (34K):
[in this window]
[in a new window]

  		Figure 1. Electron micrographs of mitochondria isolated from rat forebrain. Mitochondria displayed elongated (A) or round (B) and had numerous transverse cristae with a regular pattern with an electron-dense intramitochondrial matrix with no signs of obvious damage. The outer and inner mitochondrial membranes were clearly identifiable. Few cell debris were visible as diffuse dark material and small vesicles. Bar: 200 nm.

 

Changes of Absorbance in Mitochondria Induced by Ca2+
As shown in Figure 2, the exposition of isolated mitochondria to Ca2+ (40–200 µM) induced a dose-dependent increase in mitochondrial swelling, as detected by a decrease of absorbance (Figs. 2A–C). This process was classically inhibited by 5 min preincubation with 5 µM cyclosporine A (a pharmacological inhibitor of PTP) (Fig. 2B), which confirmed that Ca2+-induced mitochondrial swelling was due to opening of the PTP. In all three concentrations, Ca2+-induced mitochondrial swelling was reduced in the 1.0 MAC and 1.5 MAC desflurane groups compared with 1.5 MAC halothane group. The effect was significant and persisted for at least 10 min after the addition of Ca2+. Figure 2D shows the average change of absorbance ([Delta]A) of 100 µM Ca2+ in all groups. The changes of [Delta]A in the 1.5 MAC halothane group, 1.0 MAC and 1.5 MAC desflurane groups were higher by 55.5% (P < 0.001), 18.4% (P = 0.045), and 19.5% (P = 0.031) compared with the sham group, respectively. In the 1.0 MAC and 1.5 MAC desflurane groups, these changes were reduced by 23.9% (P < 0.001) and 23.2% (P < 0.001) compared with the 1.5 MAC halothane group, respectively. No significant differences were found between the 1.0 MAC and 1.5 MAC desflurane groups.


Figure 232
View larger version (30K):
[in this window]
[in a new window]

  		Figure 2. Desflurane reduces Ca2+-reduced swelling in brain mitochondria. Mitochondria were isolated from rat forebrain by the method mentioned above. Ca2+ was added to a suspension containing an aliquot of mitochondria, and mitochondria swelling was quantified by measuring the reduction of absorbance at 540 nm wavelength. Typical representative traces from experiments are shown for brain mitochondria isolated from sham, 1.5 MAC halothane, 1.0 MAC desflurane, and 1.5 MAC desflurane groups. Effect of 40 (A), 100 (B), and 200 (C) µM Ca2+ on A540 in mitochondria from sham, 1.5 MAC halothane- and desflurane-treated groups are shown. Pore inhibitor cyclosporin A 5 µM abolished the effects on A540 in both groups, confirming that Ca2+-induced mitochondrial swelling is due to permeability transition pore. The average change in A540 over 10 min of Ca2+-induced mitochondrial swelling is indicated at 100 µM Ca2+ (D). Each bar represents the mean ± sd. *P < 0.05 versus sham group, #P < 0.05 versus 1.5 MAC halothane group. CsA = Cyclosporin A.

 

Effect of Desflurane on MMP
Figures 3A and B show the changes of MMP in all groups. The addition of 40 µM Ca2+ to the isolated mitochondria induced a progressive increase of fluorescence intensity because of the discharge of Rh123 from the mitochondria, which indicated a loss of MMP. The changes of fluorescent intensity of the mitochondrial suspension in the 1.5 MAC halothane group increased significantly (31.5%, P = 0.007) 5 min after the addition of Ca2+ compared with the sham group. An obvious inhibition of MMP loss was observed in the 1.0 MAC and 1.5 MAC desflurane group by 22.4% (P = 0.013) and 20.4% (P = 0.027), respectively, when compared with the 1.5 MAC halothane group. However, there was no obvious difference in the extent of changes of MMP when comparing the 1.0 MAC desflurane with 1.5 MAC desflurane groups.


Figure 332
View larger version (28K):
[in this window]
[in a new window]

  		Figure 3. Effect of halothane and desflurane on Ca2+-induced dissipation of mitochondrial membrane potential expressed as arbitrary fluorescence units. Experimental conditions are described in Methods. The fluorescence intensity was determined at ex 503 nm, em 525 nm. Mitochondria (0.5 mg/mL), 40 µM Ca2+ were added where indicated by the arrows (A); results were representative of the experiment. {Delta}Change of arbitrary fluorescence units are shown in (B); values are means ± sd (n = 10). *P < 0.05 versus sham group, #P < 0.05 versus 1.5 MAC halothane group. Mit = mitochondria.

 

Effect of Desflurane on Mitochondrial Complexes
As shown in Figure 4, the activities of mitochondrial complexes I + III, II + III, and IV changed differently in the separate groups. The activities of complexes I + III and IV in the 1.5 MAC halothane group were significantly inhibited (33.7%, P = 0.001; 47.4%, P < 0.001) compared with the sham group. MAC 1.0 and 1.5 MAC desflurane inhalation enhanced the activities of complexes I + III (34.6%, P = 0.027; 38.7%, P = 0.011) and IV (53.9%, P = 0.009; 55.8%, P = 0.007) compared with the 1.5 MAC halothane group. But there was no significant change in the activity of complexes II + III in all groups. No significant differences were observed in different concentrations of desflurane on complexes I + III, II + III, and IV activities.


Figure 432
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. Activities of complexes I + III, II + III, and IV in mitochondria isolated from rat forebrain of the four experimental groups. Left ordinate represents data from complex I + III and II + III; right ordinate represents data from complex IV. Enzyme activities are expressed as mean ± sd for each group (n = 10). *P < 0.05 when compared with sham group. #P < 0.05 when compared with 1.5 MAC halothane group. NCCR = complexes I + III; SCCR = complexes II + III; CCO = complex IV.

 

DISCUSSION

The major finding of the current study is that desflurane seems to induce tolerance and improve the function of mitochondria in forebrain ischemia reperfusion rats when compared with halothane. We show here that mitochondrial dysfunction is ameliorated by desflurane treatment, indicated by the inhibition of mitochondria swelling, stabilization of membrane potential, and improvement of mitochondria respiratory function.

The brain is very susceptible to ischemia and hypoxia, and reperfusion after ischemia may not improve the function of cells but may actually aggravate the damage. Cerebral ischemic tolerance can be improved by ischemia preconditioning and also by drugs. Among the latter, volatile anesthetics have been considered potentially beneficial. Several studies suggested that volatile anesthetics could influence the apoptosis-regulating proteins, improve neuronal function, and reduce infarct area.5,21 Because desflurane allows rapid emergence from anesthesia, its value in neurosurgical procedures is obvious. Previous reports have shown that isoflurane and desflurane produced anesthetic preconditioning, which protected the myocardium against infarction after ischemia reperfusion in animals.22,23 Tsai et al.24 reported that desflurane caused a reduction of infarct volume and lactate dehydrogenase activity in plasma, which are indices of neural cell damage after cerebral ischemia and reperfusion injury. However, the precise mechanisms responsible for desflurane-induced protection remain unclear, and few studies have investigated the effects of desflurane on function of mitochondria after brain ischemia.

Apoptosis has been implicated in ischemic cell death.25,26 Although the exact mechanisms and pathways of apoptosis remain poorly defined, the role of mitochondria in vitro and in vivo has been extensively described.27 The PTP, which is formed at contact sites between the inner and outer mitochondrial membranes, was identified as a major factor in the mitochondrial pathways leading to cell death.28,29 We found that desflurane attenuates Ca2+-induced mitochondrial swelling, which is indicative of inhibition of the opening of PTP. This is in close agreement with other investigators who used a rabbit myocardial ischemia model.30 During reperfusion, mitochondria become re-energized, and there is uptake of cytosolic Ca2+ into mitochondria, gradual recovery of pH to normal, and excessive free-radical generation, which favor the opening of PTP.31,32 This would cause matrix swelling, collapse of the inner membrane potential, uncoupling of the respiratory chain, efflux of Ca2+, release of small proteins,1,33–35 and initiation of the process of DNA fragmentation and apoptosis.36,37 Therefore, it is possible that the protective effects of desflurane on ischemic reperfusion injury are directly or indirectly via modulating mitochondria PTP. MMP, which is generated by the mitochondrial electron transport chain, is an important variable for mitochondrial functionality and an indirect indicator of energy status of the cell. Maintenance of the MMP is necessary for the mitochondria to perform their functions, while dissipation of MMP seems to be a consequence of severe energy deficit, leading to necrosis and apoptosis.38 This study showed 1.0 MAC and 1.5 MAC desflurane significantly prevented the collapse of MMP induced by ischemia and reperfusion when compared with 1.5 MAC halothane, which confirmed the hypothesis that desflurane inhibited the opening of mitochondrial PTP.

The activities of the mitochondrial respiratory chain enzymes complexes measured in our research suggest that ischemia reperfusion injury damages the function of mitochondrial enzymes complexes. This is in agreement with previous studies.39,40 Recent investigations have shown that oxidative stress is implicated in the pathophysiologic changes that ensue after dysfunction of the respiratory chain in cerebral ischemia.41 The generation of oxidant species is an important event during cerebral ischemia, which provokes damage to lipids, DNA, and proteins, leading to neuronal death.42,43 The bursting of Formula and H2O2 produced by the flavin mononucleotide group of complex I and the ubiquinone-cytochrome b-c1 region of complex III during the reperfusion period can damage the mitochondria respiratory chain, which influences the generation of adenosine triphosphate by oxidative phosphorylation and produces more free radicals.44,45 We also demonstrated in the current study that 1.0 MAC and 1.5 MAC desflurane could improve the function of mitochondria respiratory chain enzyme complexes, which may result in a decrease of oxidant species production and protection of oxidative phosphorylation during reperfusion after ischemia, thereby ameliorating ischemia reperfusion injury.

We found that the 1.0 MAC and 1.5 MAC desflurane groups induced more protection than the 1.5 MAC halothane group in the function of mitochondria; however, there were no significant differences between the 1.0 MAC and 1.5 MAC desflurane groups. This is interesting because although the 1.5 MAC desflurane had a greater depth of anesthesia than the 1.0 MAC desflurane, greater protection of mitochondria was not observed. However, this is similar to the finding of Wise-Faberowski et al. in their ex vivo work.46 Thus, the greater neuroprotection of desflurane could not be attributed to the difference of the administered anesthetic concentrations. It is possible that there is a ceiling effect in desflurane’s brain protection effect. Further studies in animals to confirm the possible differential effects on function of mitochondria and neuronal survival among different doses of desflurane are needed.

The limitation of this study must be clarified. Although we demonstrated that desflurane reduced Ca2+-induced swelling in isolated mitochondria, whether this effect was direct or indirect was not confirmed. Further study of the pathway of this effect is needed.

In conclusion, we found that desflurane shows better preservation on the functions of mitochondria observed at 4 h after cerebral ischemia reperfusion injury, indicated by the inhibition of mitochondrial swelling, maintenance of membrane potential, and improvement in the function of mitochondria respiratory complexes I + III and IV when compared with halothane. However, no additional protection was observed at the higher dose of desflurane.

ACKNOWLEDGMENTS

The author thank Peng Pan, MD, PhD, Xianfeng Ren, MD, PhD, and Yue Ma (Technician, Third Affiliated Hospital of Harbin Medical University, China) for their support and technical assistance, and professor Tsutomu Kobayashi for reviewing the manuscript.

Footnotes

Accepted for publication November 27, 2007.

Supported by Ministry of Major Science & Technology of Heilongjiang, Heilongjiang, China.

REFERENCES

  1. Martinou JC, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001;2:63–7[Web of Science][Medline]
  2. Desagher S, Martinou JC. Mitochondria as the central control point of apoptosis. Trends Cell Biol 2000;10:369–77[Web of Science][Medline]
  3. Yoshida H, Kong YY, Yoshida R, Elia AJ, Hakem A, Hakem R, Penninger JM, Mak TW. Apaf1 is required for mitochondria pathways of apoptosis and brain development. Cell 1998;94:739–50[Web of Science][Medline]
  4. Karbowski M, Kurono C, Wozniak M, Ostrowski M, Teranishi M, Nishizawa Y, Usukura J, Soji T, Wakabayashi T. Free radical-induced megamitochondria formation and apoptosis. Free radical Biol Med 1999;26:396–409[Web of Science][Medline]
  5. Warner DS, Ludwig PS, Pearlstein R, Brinkhous AD. Halothane reduces focal ischemic injury in the rat when brain temperature is controlled. Anesthesiology 1995;82:1237–45[Web of Science][Medline]
  6. Hoffman WE, Thomas C, Albrecht RF. The effect of halothane and isoflurane on neurologic outcome following incomplete cerebral ischemia in the rat. Anesth Analg 1993;76:279–83[Web of Science][Medline]
  7. Haelewyn B, Yvon A, Hanouz JL, MacKenzie ET, Ducouret P, Gerard JL, Roussel S. Desflurane affords greater protection than halothane against focal cerebral ischaemia in the rat. Br J Anaesth 2003;91:390–6[Abstract/Free Full Text]
  8. Eger EI II, Gong D, Koblin DD, Bowland T, Ionescu P, Laster MJ, Weiskopf RB. The effect of anesthetic duration on kinetic and recovery characteristics of desflurane versus sevoflurane, and on the kinetic characteristics of compound A, in volunteers. Anesth Analg 1998;86:414–21[Abstract]
  9. Englelhard K, Werner C, Reeker W, Lu H, Mollenberg O, Mielke L, Kochs E. Desflurane and isoflurane improve neurological outcome after incomplete cerebral ischaemia in rats. Br J Anaesth 1999;83:415–21[Abstract/Free Full Text]
  10. Hoffman WE, Charbel FT, Edelman G, Ausman JI. Thiopental and desflurane treatment for brain protection. Neurosurgery 1998;43:1050–3[Web of Science][Medline]
  11. Busto R, Dietrich WD, Globus MY, Ginsberg MD. The importance of brain temperature in cerebral ischemic injury. Stroke 1989;20:1113–4[Free Full Text]
  12. Perez-Pinzon MA, Basit A, Dave KR, Busto R, Veauvy C, Saul I, Ginsberg MD, Sick TJ. Effect of the first window of ischemic preconditioning on mitochondrial dysfunction following global cerebral ischemia. Mitochondrion 2002;2:181–9[Web of Science][Medline]
  13. Kristian T, Weatherby TM, Bates TE, Fiskum G. Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria. J Neurochem 2002;83:1297–308[Web of Science][Medline]
  14. Schroeter H, Boyd CS, Ahmed R, Spencer JP, Duncan RF, Rice-Evans C, Cadenas E. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis. Biochem J 2003;372:359–69[Web of Science][Medline]
  15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54[Web of Science][Medline]
  16. Kristian T, Gertsch J, Bates TE, Siesjo BK. Characteristics of the calcium-triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: effect of cyclosporin a and ubiquinone o. J Neurochem 2000;74:1999–2009[Web of Science][Medline]
  17. Galindo MF, Jordan J, Gonzalez-Garcia C, Cena V. Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria. Br J Pharmacol 2003;139:797–804[Web of Science][Medline]
  18. Emaus RK, Grunwald R, Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 1986;850:436–48[Medline]
  19. Chuang YC, Tsai JL, Chang AY, Chan JY, Liou CW, Chan SH. Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat. J Biomed Sci 2002;9:542–8[Web of Science][Medline]
  20. Brusque AM, Borba Rosa R, Schuck PF, Dalcin KB, Ribeiro CA, Silva CG, Wannmacher CM, Dutra-Filho CS, Wyse AT, Briones P, Wajner M. Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid. Neurochem Int 2002;40:593–601[Web of Science][Medline]
  21. Englelhard K, Werner C, Eberspacher E, Pape M, Blobner M, Hutzler P, Kochs E. Sevoflurane and propofol influence the expression of apoptosis-regulating proteins after cerebral ischaemia and reperfusion in rats. Eur J Anaesthesiol 2004;21:530–7[Web of Science][Medline]
  22. Kersten JR, Schmeling TJ, Pagel PS, Gross GJ, Warltier DC. Isoflurane mimics ischemic preconditioning via activation of K(ATP) channels: reduction of myocardial infarct size with an acute memory phase. Anesthesiology 1997;87:361–70[Web of Science][Medline]
  23. Tsai SK, Lin SM, Huang CH, Hung WC, Chih CL, Huang SS. Effect of desflurane-induced preconditioning following ischemia-reperfusion on nitric oxide release in rabbits. Life Sci 2004;76:651–60[Web of Science][Medline]
  24. Tsai SK, Lin SM, Hung WC, Mok MS, Chih CL, Huang SS. The effect of desflurane on ameliorating cerebral infarction in rats subjected to focal cerebral ischemia-reperfusion injury. Life Sci 2004;74:2541–9[Web of Science][Medline]
  25. Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting a role for programmed cell death in focal cerebral ischemia in rats. Stroke 1993;24:2002–8[Abstract/Free Full Text]
  26. Charriaut-Marlangue C, Margaill I, Represa A, Popovici T, Plotkine M, Ben-Ari Y. Apoptosis and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis. J Cereb Blood Flow Metab 1996;16:186–94[Web of Science][Medline]
  27. Solenski NJ, diPierro CG, Trimmer PA, Kwan AL, Helm GA. Ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia. Stroke 2002;33:816–24[Abstract/Free Full Text]
  28. Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F. Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem 1999;264:687–701[Web of Science][Medline]
  29. Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J 1999;341:233–49[Web of Science][Medline]
  30. Piriou V, Chiari P, Gateau-Roesch O, Argaud L, Muntean D, Salles D, Loufouat J, Gueugniaud PY, Lehot JJ, Ovize M. Desflurane-induced preconditioning alters calcium-induced mitochondrial permeability transition. Anesthesiology. 2004;100:581–8[Web of Science][Medline]
  31. Back T, Hoehn M, Mies G, Busch E, Schmitz B, Kohno K, Hossmann KA. Penumbral tissue alkalosis in focal cerebral ischemia: relationship to energy metabolism, blood flow, and steady potential. Ann Neurol 2000;47:485–92[Web of Science][Medline]
  32. Piantadosi CA, Zhang J. Mitochondrial generation of reactive oxygen species after brain ischemia in the rat. Stroke 1996; 27:327–31[Abstract/Free Full Text]
  33. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001;15:2922–33[Free Full Text]
  34. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–12[Abstract/Free Full Text]
  35. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996;86:147–57[Web of Science][Medline]
  36. Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 1998;391:96–9[Medline]
  37. Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 1998;391:43–50[Medline]
  38. Iijima T. Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 2006;55:234–43[Web of Science][Medline]
  39. Allen KL, Almeida A, Bates TE, Clark JB. Changes of respiratory chain activity in mitochondrial and synaptosomal fractions isolated from the gerbil brain after graded ischemia. J Neurochem 1995;64:2222–9[Web of Science][Medline]
  40. Almeida A, Allen KL, Bates TE, Clark JB. Effect of reperfusion following cerebral ischemia on the activity of the mitochondrial respiratory chain in the gerbil brain. J Neurochem 1995;65:1698–703[Web of Science][Medline]
  41. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000;7:153–63[Medline]
  42. Chan PH. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res 2004;29:1943–9[Web of Science][Medline]
  43. Christophe M, Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia-reperfusion. Curr Pharm Des 2006;12:739–757[Web of Science][Medline]
  44. Liu Y, Fiskum G, Schubert D. Generation of reactive oxygen species by the mitochondrial electron transport chain. J Neurochem 2002;80:780–7[Web of Science][Medline]
  45. Han D, Williams E, Cadenas E. Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. Biochem J 2001;353:411–6[Web of Science][Medline]
  46. Wise-Faberowski L, Raizada MK, Sumners C. Desflurane and sevoflurane attenuate oxygen and glucose deprivation-induced neuronal cell death. J Neurosurg Anesthesiol 2003;15:193–9[Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, B.
Right arrow Articles by Li, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, B.
Right arrow Articles by Li, W.
Related Collections
Right arrow Mechanisms
Right arrow Neuroanesthesia
Right arrow Preclinical Pharmacology
Right arrow Pharmacology

Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press