adhesion molecules such as Mac-1 (cluster of differen- tiation molecule 11b/18) .... 0.1 nmol/mg protein), although no significant differences were noticed (data.
Effects of Hyperbaric Oxygen Exposure on Experimental Hepatic Ischemia Reperfusion Injury: Relationship Between Its Timing and Neutrophil Sequestration Kenji Kihara, Shinichi Ueno, Masahiko Sakoda, and Takashi Aikou Recent studies have shown that hyperbaric oxygen therapy (HBOT) reduces neutrophil endothelial adherence in venules and also blocks the progressive arteriolar vasoconstriction associated with ischemia-reperfusion (I-R) injury in the extremities and the brain. In order to elucidate the effects of HBOT after I-R in digestive organs, particularly in the liver, we evaluated the following: 1) the relationship between timing of HBOT and tissue damage; and 2) HBOT’s effects on neutrophil sequestration. Using a hepatic I-R (45 minute) model in male rats, survival rate, liver tissue damage, and neutrophil accumulation within the sinusoids in the HBOT-treated group (Group H) were compared to those in the nontreated group (Group C). For the HBOT-treated group, HBOT was administered as 100% oxygen, at 2.5 atm absolute, for 60 minutes. When HBOT was given 30 minute after I-R, the survival rate was much better in Group H than in Group C. HBOT performed within 3 hours of I-R markedly suppressed increases in the malondialdehyde level in tissues of the liver and lessened the congestion in the sinusoids. In addition, HBOT just after I-R caused decreased number of cells stained by the naphthol AS-D chloroacetate esterase infiltrating into the sinusoids. HBOT 3 hours after reperfusion, however, showed no clear effects upon neutrophil sequestration compared to Group C. These results indicate that HBOT performed within 3 hours of I-R alleviates hepatic dysfunction and improves the survival rate after I-R. Herein, we propose 1 possible mechanism for these beneficial effects: early HBOT given before neutrophil-mediated injury phase may suppress the accumulation of neutrophils after I-R. In conclusion, we believe that the present study should lead to an improved under-
Abbreviations: HBOT, hyperbaric oxygen therapy; I-R, ischemia-reperfusion; MDA, malondialdehyde; TNF-␣, tumor necrosis factor-a; IL, interleukin; ICAM, intracellular adhesion molecule-1. From the Department of Surgical Oncology and Digestive Surgery, Kagoshima University Graduate School of Medicine and Dental Sciences, Kagoshima, Japan. Received January 24, 2005; accepted June 13, 2005. Address reprint requests to Shinichi Ueno, Department of Surgical Oncology and Digestive Surgery, Field of Oncology, Course of Advanced Therapeutics, Kagoshima University Graduate School of Medicine and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890, Japan. Telephone: 81-99-275-5361; FAX: 81-99-265-7426; E-mail: ueno1@m. kufm.kagoshima-u.ac.jp Copyright © 2005 by the American Association for the Study of Liver Diseases Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/lt.20533
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standing of HBOT’s potential role in hepatic surgery. (Liver Transpl 2005;11:1574-1580.)
W
arm hepatic ischemia-reperfusion (I-R) is characterized by circulatory and metabolic disruptions, liver dysfunction, and tissue damage. Recent advances in the research regarding the basic mechanisms of I-R showed the significance of Kupffer cells and neutrophils.1 Kupffer cells play a central role as the initial cytotoxic cell type and as a source of many proinflammatory mediators such as tumor necrosis factor-␣ (TNF-␣) and interleukin (IL)-6.2-6 Subsequently, neutrophils are activated and migrate into the liver. Researchers have pointed out that accumulated neutrophils contribute to the injury several hours after the initiation of reperfusion.7 In fact, antibodies against adhesion molecules such as Mac-1 (cluster of differentiation molecule 11b/18) or intracellular adhesion molecule-1 (ICAM-1) are still beneficial, ever if administered during the Kupffer cell-mediated injury phase.7-12 Hyperbaric oxygen therapy (HBOT) is a specific type of oxygen administration that aims to improve numerous kinds of hypoxic disorders by increasing the amount of dissolved oxygen within the blood.13 Recent studies have shown that HBOT reduced neutrophil endothelial adherence in venules and also blocked the progressive arteriolar vasoconstriction associated with reperfusion injury.14 Using a rat septic model, we also showed that HBOT could modify the nuclear factor-kB activation in the intestinal mucosa and attenuate the sequential nitric oxide overproduction and myeloperoxidase activation.15 In terms of clinical effects, there are reports of favorable effects of HBOT after I-R in the extremities and the brain.16-20 As for the effect of HBOT after I-R in digestive organs, particularly in the liver, using hepatic I-R model in rats, only Chen et al.21 reported that HBOT before ischemia significantly depressed the tissue malondialdehyde (MDA) level and effectively preserved the tissue adenosine triphosphate level compared to the ischemia control group. As for the mechanism of these benefits, they showed that hyperbaric oxygen pretreatment significantly less-
Liver Transplantation, Vol 11, No 12 (December), 2005: pp 1574-1580
Hyperbaric Oxygen Therapy and Liver Ischemia
Figure 1. Experimental design.
ened adherent leukocyte count and improved flow velocity in postsinusoidal venules. Buras et al.22 also showed that the beneficial effects of HBOT in reperfusion injury may be mediated through decreased expression of ICAM-1 in an in vitro endothelial model of I-R injury. Some researchers, however, reported that HBOT did not alter the expression of adhesion molecules (e.g., cluster of differentiation molecule 18) on neutrophils.12,23 It is not fully understood whether HBOT itself modifies the neutrophil tissue accumulation in vivo. Thus, the primary purposes of this study were 1) to evaluate the effects of HBOT, particularly when performed for I-R in hepatic tissue and 2) to determine whether HBOT could modify the sequestration of neutrophils.
Materials and Methods Experimental Model
Experiments and Parameters
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water freely were used. The animals were housed in compliance with good laboratory practice and maintained for the protection of experimental animals (constant temperature: 22 ⫾ 2°C; relative humidity: 55 ⫾ 10%, through the study). Each rat was anesthetized with an intraperitoneal injection of pentobarbital sodium (nembutal, 50mg/ mL). An abdominal midline incision was made and the hepatoduodenal ligament was clamped with a microvascular clamp (Sugita Standard aneurysm clip; Mizuho Medical, Tokyo) in order to produce total hepatic ischemia. Forty-five minutes later, reperfusion of the liver was initiated by removal of the clamp. The incision was sutured just after the initiation of reperfusion. For HBOT-treated groups, HBOT at 2.5 atm with inhalation of 100% oxygen, was performed for 60 minutes. Most rats recovered their consciousness around 20 minutes after the skin-closure, and thus, some recovered in the chamber of HBOT started at just after I-R. The protocol was approved by the University of Medicine of Kagoshima Animal Care Committee.
Assay of Lipid Peroxidation in Liver Tissue Lipid peroxidation in the liver was determined by measuring the levels of MDA, which is an end product of lipid metabolism. The liver was flushed with ice-cold 0.9% NaCl via the portal vein and rapidly excised. A portion of the liver was removed and immediately frozen and stored at -80°C until homogenization. The homogenizations were performed after combining a ratio of 0.5 gm of wet tissue with 5 mL of 50 mmol/L phosphate buffer (pH 7.4) (homogenizer; Iuchi Seieidou, Osaka, Japan). The content of MDA in the homogenate was determined using a colorimetric reaction with thiobarbituric acid, as described by Bieri and Anderson.24 The protein concentration was calculated according to the method of Bradford25 by using Bio-Rad Protein Assay Kits (Bio-Rad Laboratories, Tokyo, Japan).
Histological Studies
Light Microscopy
We investigated 3 aspects of I-R injury and the impact of HBOT, as shown in Figure 1. Experiment 1: survival rate 48 hours following reperfusion; experiment 2: the relationship between the timing of HBOT and tissue damage; and experiment 3: impact of HBOT on neutrophil sequestration. In experiment 2, the timing of HBOT was investigated; we analyzed lipid peroxidation activity by the thiobarbituric acid method and observed tissue damage microscopically. In experiment 3, we used naphthol AS-D chloroacetate staining for counting of neutrophils in the sinusoids.
Infiltration of neutrophils. Hepatic specimens for light microscopy were fixed in 10% formalin and then bedded in paraffin, and 4-mm-thick sections were cut. Polymorphonuclear cells were stained by using the naphthol AS-D chloroacetate esterase technique (Sigma Chemical, St. Louis, MO)26 to investigate the accumulation of polymorphonuclear cells in the liver after reperfusion. Polymorphonuclear cells were identified by positive staining and morphology and counted under 50 high-power fields of a light microscope. Double blind analyses were performed on all samples in the histological studies.
Total Hepatic Ischemia Model
Scoring of Histological Changes
Male Wister rats weighing 230 to 280 gm that were fasted for 12 hours before the experiments but allowed to drink
Sections were stained with hematoxylin and eosin for histological examination. Blind analysis was performed on all sam-
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Table 1. Survival Rate 48 Hours Following Reperfusion Survival after I-R†
Control HBOT
n
24 hours
48 hours
12 12
6 (50.0) 10 (83.3)
5 (41.4) 10 (83.3)*
Abbreviation I-R, ischemia-reperfusion. *P ⬍ 0.05 vs. CONTROL †The number in parentheses shows survival rate.
ples. Histological changes were scored from 0 to 4 based upon the degree of cytoplasmic vacuolization, sinusoidal congestion, and necrosis of parenchymal cells, according to the criteria of Suzuki et al.27
Electron Microscopy For liver tissue preparation for scanning electron microscopy, animals were perfused via the heart, first with 0.1 m/L phosphate buffer (pH 7.4) and then with 0.1 m/L phosphate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde. After perfusion fixation, small tissue blocks were excised and fixed with the same solution at 4°C overnight followed by postfixation with 1% OsO4 at 4°C for 1 hour. Samples were then washed with phosphate buffer, dehydrated in a graded series of ethanol and cracked in ethanol frozen by liquid nitrogen. After conductive staining with 2% tannic acid and 1% OsO4, samples were immersed into t-butyl alcohol and freeze-dried using a freeze drying apparatus (ID-2; Eiko Engineering, Tokyo, Japan). The dried samples were coated with gold by means of an ion sputter (FC-1100; Japan Electron Optics Laboratory, Tokyo, Japan), and were examined under a scanning electron microscope (S-800; Hitachi, Tokyo, Japan).
Relationship Between Timing of HBOT and Hepatic Tissue Damage (Experiment 2)
Lipid Peroxidation of the Liver Tissue First, we examined the significance of HBOT just after reperfusion. No significant difference was seen between the groups in the MDA level 1 hour after reperfusion. However, the MDA level 4 hours after reperfusion was clearly increased in Group C compared to that noted in Group H (P ⬍ 0.01; Fig. 2A). In addition, the effects of HBOT 3 or 6 hours after reperfusion were examined. HBOT 3 hours after reperfusion decreased the MDA level (P ⬍ 0.01); however, HBOT 6 hours after reperfusion did not prevent tissue damage (Fig. 2B and C). Liver tissue MDA level 4 hours after the sham operation was slightly increased in rats given HBOT (n ⫽ 3; 0.3 ⫾ 0.2 nmol/mg protein) compared to those not given HBOT (n ⫽ 3; 0.2 ⫾ 0.1 nmol/mg protein), although no significant differences were noticed (data not shown).
Scoring of Histological Changes In Group C, the scores of the 3 histologic parameters, in particular, necrosis of parenchymal cells, increased over time (see Fig. 3 and Table 2). When HBOT was performed immediately after reperfusion, there was a numerical decrease in all histologic parameters, although significant differences were noted only in the degree of sinusoidal congestion.
Statistical Analysis Twelve animals per group were used for experiment 1, and 5 to 7 animals per group at each time point were used for experiments 2 and 3. All data are expressed as means ⫾ standard deviation. Statistical comparisons were analyzed with unpaired Student’s t-test, Mann-Whitney U-test, and 2-test, as appropriate. Significance was assumed at P values ⬍ 0.05.
Results Survival Rate (Experiment 1) Table 1 shows the survival rates following reperfusion. In Group H, HBOT was given at 30 minutes after reperfusion. The survival rate was much better in Group H than in Group C (P ⬍ 0.05).
Figure 2. The change of liver tissue thiobarbituric acid reactive substances (TBARS) level. HBOT was given immediately (A), 3 hours (B), and 6 hours (C) after I-R. *P < 0.01 vs. Group H.
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Hyperbaric Oxygen Therapy and Liver Ischemia
Figure 3. Microscopic findings of liver tissue (H&E, ⴛ200). (A and B) 4 and 7 hours after I-R in Group C (non-HBOT); (C and D) 4 and 7 hours after I-R in Group H; HBOT was performed immediately after I-R.
Electron Microscopy Changes Figure 4 shows the microstructures of the liver that underwent HBOT just after I-R and were excised 7 hours after I-R. The structure of the sinusoidal endothelium remained almost undisturbed in Group H, although sinusoidal endothelial cells were swollen. On the other hand, more severe changes in the sinusoids, including sinusoidal denudation, were noted in Group C. Neutrophil Accumulation (Experiment 3) The number of neutrophils in the sinusoids after I-R was shown in Figure 5. When HBOT was performed immediately after reperfusion, there was no difference in the number of neutrophils in the sinusoids examined 1 hour after reperfusion. Three hours later, the number of neutrophils was significantly lower in Group H than in Group C (P ⬍ 0.05). Neutrophil count in the sinusoids 4 hours after the sham operation was not different between rats with (75 ⫾ 20) or without HBOT (68 ⫾ 18) (data not shown). On the other hand, HBOT 3
hours after reperfusion did not affect the degree of neutrophil accumulation compared to the control group.
Discussion We undertook this study to examine whether HBOT diminished tissue injury in the liver after ischemiareperfusion. Furthermore, if HBOT were found to have such a beneficial effect, we desired to elucidate the mechanism by which HBOT achieves its effect, while paying special attention to the relationship between suppression of neutrophil tissue accumulation and the dysfunction of the liver after I-R. The findings of experiment 1 demonstrated that HBOT performed 30 minutes after I-R improved the survival rate of the experimental animals. The findings in experiment 2 indicated that HBOT performed within 3 hours of I-R markedly suppressed increases in the MDA level in tissues of the liver. Histological examinations revealed that the congestion in the sinusoids was less marked in the experiment 2 HBOT group than in the nontreated group.
Table 2. Histological Studies. 4 hours after I-R
7 hours after I-R
Analysis
Group C
Group H
Group C
Group H
Cytoplasmic vacuolization Sinusoidal congestion Necrosis of parencymal cells
1.5 ⫾ 1.0 1.6 ⫾ 0.9 1.0 ⫾ 0.9
1.1 ⫾ 0.5 1.0 ⫾ 0.8* 0.8 ⫾ 0.7
1.8 ⫾ 1.5 1.7 ⫾ 1.2 2.3 ⫾ 0.5†
1.4 ⫾ 1.1 1.1 ⫾ 0.6* 1.7 ⫾ 0.5*
NOTE. Histological changes 4 and 7 hours after I-R for both groups were scored from 0 to 4 based on the degree of cytoplasmic vacuolization, sinusoidal congestion, and necrosis of parenchymal cells. For Group H, HBOT was given just after I-R. Abbreviation: I-R, ischemia-reperfusion. Data are expressed as means ⫾ Standard deviation. *P ⬍ 0.05 vs. Group C. †P ⬍ 0.05 vs. 4 hours after I-R between Group C.
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Figure 4. Electron microscopic findings (ⴛ9,000) 7 hours after reperfusion. HBOT was performed just after I-R. (A) Non-HBOT-treated group; (B) HBOT-treated group.
Electron microscope examinations showed that swelling and disruption of sinusoidal endothelial cells were reduced in the HBOT group compared to the nontreated group. Thus, the above findings indicate that HBOT has a beneficial effect on I-R-induced damages in the liver, as has been observed in other models.28-30 Our results agree with those reported by Chen et al.21 in that HBOT performed before I-R suppressed disruption of the tissues as indicated by improved levels of MDA and ATP. Of significance, our study shows that HBOT is effective up to 3 hours after I-R. Regarding the effect of HBOT on I-R-induced damages, some researchers have speculated that the altered microcirculatory environment after HBOT changed the response to I-R by inactivating free radical generation in parenchymal cells and endothelial tissue.14 Furthermore, Chen et al.21 evaluated dynamic changes in microvessel flow and leukocyte endothelial adherence during reperfusion by using an in vivo microscope and indicated that pretreated-HBOT lessened the adherent leukocyte count in the sinusoids and postsinusoidal venules. We have examined the effect of HBOT on I-R-induced damages with close attention to suppression of the accu-
mulation of neutrophils after I-R. The direct cause of tissue dysfunction after I-R is considered to be the accumulation of neutrophils, adhesion of neutrophils to endothelial cells, and excessive production of reactive oxygen species, all of which ensue from the generation of reactive oxygen species.31 The key to I-R therapy, therefore, is 1) to reduce the generation of inflammatory mediators (C5a, TNF-␣, platelet activity factor, and C-X-C chemokine), which cause the accumulation of neutrophils, and also 2) to reduce adhesion molecules in neutrophils (e.g., Mac-1 and LFA-1) and those in the vessel endothelium (e.g., ICAM-1). It has been shown that TNF-␣, reactive oxygen species produced by Kupffer cells, and inflammatory mediators released from neutrophils (which infiltrated during reperfusion) all contribute effectively to the generation of the hepatic dysfunction after I-R.32 Neutrophils are recruited and activated by chemokines such as IL-8 whose production in hepatocytes and sinusoidal endothelial cells is most effectively upregulated during ischemia.33 It seems possible that HBOT increases the concentration of oxygen that can be dissolved in the tissue, alleviates the oxygen deficiency induced by ischemia, and thereby suppresses the production of chemokines. This conjecture may be supported by our finding that only HBOT performed immediately after I-R was able to suppress the expression of cluster of differentiation molecule 18 in neutrophils (data not shown). In fact, we observed previously that HBOT immediately after hepatectomy suppressed increases in blood concentrations of interleukin-8 and polymorphonuclear leukocyte elastase in hepatic veins,34 although this observation should be reexamined experimentally in the future. An important point to emphasize is the fact that only when HBOT is performed before the neutrophil-mediated injury phase (i.e., during the Kupffer cell-mediated injury phase) is it
Figure 5. Influence of HBOT upon neutrophil accumulation in the sinusoids. HBOT was given immediately following or 3 hours after reperfusion. *P < 0.01 between the groups.
Hyperbaric Oxygen Therapy and Liver Ischemia
likely to suppress neutrophil activation. Further research should investigate whether HBOT has any direct influence upon Kupffer cells, because using an experimental shock model, Yamashita and Yamashita35 and Luongo et al.36 reported that HBOT could decrease serum Kupffer cell-mediated cytokine levels (e.g., tumor necrosis factor-a and interleukin-6) and following NOx concentration, leading to neutrophils activation. HBOT performed 3 hours after I-R was found to have no direct effect upon the accumulation of neutrophils. The results of the experiment 2 indicate, however, that even HBOT made with such a delay reduced damage to tissues, as shown by MDA level in Figure 2. As suggested by other authors, therefore, early HBOT may have another beneficial effect against I-R injury (e.g., suppression of the progressive arteriolar vasoconstriction associated with reperfusion injury),12,14,21,34 thereby preventing disruption of the microcirculation. In addition, regarding the beneficial effects of HBOT after I-R, Mrsic-Pelcic et al.37 showed that HBOT could enhance superoxide dismutase activity and preserve Na(⫹), K(⫹)-adenosine triphosphatase activity within particular periods of reperfusion in cerebral system, and Tjarnstrom et al.38 observed that HBOT stimulated a release of fibrinolytic factors such as tissue plasminogen activator and urokinase plasminogen activator in cultured endothelial cells exposed to anoxia and suggested that HBOT might prevent thrombosis and/or microembolization following I-R. Mazariegos et al.39 in Pittsburgh transplantation center also reported that HBOT could prolong the interval to retransplantation, potentially by hastening the development of hepatic artery collaterals, in patients with hepatic artery thrombosis after liver transplantation. In summary, the results of this study show that HBOT performed within 3 hours of I-R alleviates hepatic dysfunctions and improves the survival rate after I-R. As one possible mechanism, we propose that HBOT given before neutrophil-mediated injury phase suppresses the accumulation neutrophils after I-R. The beneficial effect of HBOT on hepatic failure after hepatectomy and on hyperbilirubinemia40 as well as the facilitative action of HBOT on liver regeneration have been reported previously.41 We believe that the present study supports further studies in the application of HBOT in hepatic surgery.
Acknowledgment We thank Ms. Nobue Uto at Kagoshima University (Department of Laboratory Medicine) for her assistance.
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References 1. Jaeschke H. Mechanisms of reperfusion injury after warm ischemia of the liver. J Hepatobiliary Pancreat Surg 1998;5:402 – 408. 2. Shibuya H, Ohkohchi N, Tsukamoto S, Satomi S. Tumor necrosis factor-induced, superoxide-mediated neutrophil accumulation in cold ischemic/reperfused rat liver. Hepatology 1997; 26:113-120. 3. Hanazaki K, Monma T, Hiraguri M, Ohmoto Y, Kajikawa S, Matsushita A, et al. Cytokine response to human liver ischemiareperfusion injury during hepatectomy: marker of injury or surgical stress? Hepatogastroenterology 2001;48:188-192. 4. Jaeschke H, Farhood A, Bautista AP, Spolarics Z, Spitzer JJ. Complement activates Kupffer cells and neutrophils during reperfusion after hepatic ischemia. Am J Physiol 1993;264: G801-809. 5. Andus T, Bauer J, Gerok W. Effects of cytokines on the liver. Hepatology 1991;13:364-375. 6. Liu P, Xu B, Spokas E, Lai PS, Wong PY. Role of endogenous nitric oxide in TNF-alpha and IL-1beta generation in hepatic ischemia-reperfusion. Shock 2000;13:217-223. 7. Marubayashi S, Oshiro Y, Maeda T, Fukuma K, Okada K, Hinoi T, et al. Protective effect of monoclonal antibodies to adhesion molecules on rat liver ischemia-reperfusion injury. Surgery 1997; 122:45-52. 8. Morisaki T, Goya T, Toh H, Nishihara K, Torisu M. The anti Mac-1 monoclonal antibody inhibits neutrophil sequestration in lung and liver in a septic murine model. Clin Immunol Immunopathol 1991;6: 365-375. 9. Sakamoto S, Okanoue T, Itoh Y, Sakamoto K, Nishioji K, Nakagawa, et al. Intercellular adhesion molecule-1 and CD18 are involved in neutrophil adhesion and its cytotoxicity to cultured sinusoidal endothelial cells in rats. Hepatology 1997;26:658663. 10. Jaeschke H, Farhood A, Bautista AP, Spolarcis Z, Spitzer JJ, Smith CW. Functional inactivation of neutrophils with a Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemia-reperfusion injury in rat liver. Hepatology 1993;17:915923. 11. Suzuki S, Toledo-Pereyra LH. Interleukin-1 and tumor necrosis factor production as the initial stimulants of liver ischemia and reperfusion injury. J Surg Res 1994;57:253-258. 12. Larson JL, Stephenson LL, Zamboni WA. Effect of hyperbaric oxygen on neutrophil CD18 expression. Plast Reconstr Surg 2000;105:1375-1381. 13. Boerema I, N G Meyne, W K Blummelkamp, Bouma S, Mensch MH, Kamermans F, et al. Life without blood. (A study of the influence of high atmospheric pressure and hypothermia on dilution of the blood). J Cardiovasc Surg 1960;1:133-146. 14. Zamboni WA, Roth AC, Russell RC, Graham B, Suchy H, Kucan JO. Morphologic analysis of the microcirculation during reperfusion of ischemic skeletal muscle and the effect of hyperbaric oxygen. Plast Reconstr Surg. 1993;91:1110-1123. 15. Sakoda M, Ueno S, Kihara K. A potential role of hyperbaric oxygen exposure through intestinal nuclear factor-kB. Crit Care Med 2004;32:1-8. 16. Roos JA, Jackson-Friedman C, Lyden P. Effects of hyperbaric oxygen on neurologic outcome for cerebral ischemia in rats. Acad Emerg Med 1998;5:18-24. 17. Sirsjo A, Lehr HA, Nolte D, Haapaniemi T, Lewis DH, Nylander G, Messmer K. Hyperbaric oxygen treatment
1580
18.
19. 20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Kihara et al.
enhances the recovery of blood flow and functional capillary density in postischemic striated muscle. Circ Shock 1993;40:9 – 13. Kindwall EP, Gottlieb LJ, Larson DL. Hyperbaric oxygen therapy in plastic surgery: a review article. Plast Reconstr Surg 1991; 88:898-908. Sukoff MH. Effects of hyperbaric oxygenation. J Neurosurg 2001;95:544-546. Rockswold SB, Rockswold GL, Vargo JM, Erickson CA, Sutton RL, et al. Effects of hyperbaric oxygenation therapy on cerebral metabolism and intracranial pressure in severely brain injured patients. J Neurosurg 2001;94:403-411. Chen MF, Chen HM, Ueng SW, Shyr MH. Hyperbaric oxygen pretreatment attenuates hepatic reperfusion injury. Liver 1998; 18:110-116. Buras JA, Stahl GL, Svoboda KK, Reenstra WR. Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS. Am J Physiol Cell Physiol. 2000;278:C292-C302. Hong JP, Kwon H, Chung YK, Jung SH. The effect of hyperbaric oxygen on ischemia-reperfusion: an experimental study in a rat musculocutaneous flap. Ann Plast Surg 2003;51:478-487. Bieri JG, Anderson AA. Peroxidation of lipids in tissue homogenates as related to vitamin E. Arc Biochem Biophys 1960;90: 105. Bradford MM. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. Molony WC. McPherson K. Fliegelman L. Esterase activity in leukocytes demonstrated by the use of naphtal AS-D chroloacetate substrate. J Histochem Cytochem 1960;8:200-207. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. Modulating effects of FK506 and cyclosporine. Transplantation 1993;55:1265-1272. Grundmann T, Jaehne M, Fritze G. The value of hyperbaric oxygen therapy (HBO) in treatment of problem wounds in the area of plastic-reconstructive head and neck surgery. Laryngorhinootologie 2000;79:304-310. Myers RA. Hyperbaric oxygen therapy for trauma: crush injury, compartment syndrome, and other acute traumatic peripheral ischemias. Int Anesthesiol Clin 2000;105:1375-1381. Kohshi K, Munaka M, Abe H, Tosaki T. New approaches in
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
neurosurgery and hyperbaric medicine-the importance of preventive and industrial medicine. J UOEH. 1999;21:331-339. Nardo B, Caraceni P, Pasini P, Domenicali M, Catena F, Cavallari G, et al. Increased generation of reactive oxygen species in isolated rat fatty liver during postischemic reoxygenation. Transplantation 2001;71:1816-1820. Chen KH, Reece LM, Leary JF. Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction. Free Radic Biol Med 1999;27:100-109. Yamada T, Hisanaga M, Nakajima Y, Kanehiro H, Aomatsu Y, Ko S, et al. The serum interleukin 8 level reflects hepatic mitochondrial redox state in hyperthermochemohypoxic isolated liver perfusion with use of a venovenous bypass. Surgery 1999; 125:304-314. Ueno S, Tanabe G, Kihara K, Aoki D, Arikawa K, Dogomori H, Aikou T. Early post-operative hyperbaric oxygen therapy modifies neutrophile activation. Hepatogastroenterology 1999;46: 1798-1799. Yamashita M, Yamashita M. Hyperbaric oxygen treatment attenuates cytokine induction after massive hemorrhage. Am J Physiol Endocrinol Metab 2000;278:E811-E816. Luongo C, Imperatore F, Cuzzocrea S, Filippelli A, Scafuro MA, Mangoni G, et al. Effects of hyperbaric oxygen exposure on a zymosan-induced shock model. Crit Care Med 1998;26:19721976. Mrsic-Pelcic J, Pelcic G, Vitezic D, Antoncic I, Filipovic T, Simonic A, Zupan G. Hyperbaric oxygen treatment: the influence on the hippocampal superoxide dismutase and Na⫹, K⫹-ATPase activities in global cerebral ischemia-exposed rats. Neurochem Int 2004;44:585-594. Tjarnstrom J, Holmdahl L, Falk P, Falkenberg M, Arnell P, Risberg B. Effects of hyperbaric oxygen on expression of fibrinolytic factors of human endothelium in a stimulated ischemia/ reperfusion situation. Scand J Clin Lab Invest 2001;61:539-545. Mazariegos GV, O’Toole K, Mieles LA, Dvorchik I, Meza MP, Briassoulis G, et al. Hyperbaric oxygen therapy for hepatic artery thrombosis after liver transplantation in children. Liver Transpl Surg 1999;5:429-436. Akin ML, Erenoglu C, Dal A, Erdemoglu A, Elbuken E, Batkin A. Hyperbaric oxygen prevents bacterial translocation in rats with obstructive jaundice. Dig Dis Sci 2001;46:1657-1662. Uwagawa T, Unemura Y, Yamazaki Y. Hyperbaric oxygen after portal vein embolization of the predicted remnant liver. J Surg Res 2001;100:63-68.
