Enhancement of Glucose Toxicity by HBO 127

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Tohoku J. Exp. Med., 2008, 216, 127-132

Received June 24, 2008; revision accepted for publication August 19, 2008.Correspondence: Yukita Sato, Laboratory of Biomedical Science, Department of Veterinary Medicine, College

of Bioresource Sciences, Nihon University, Fujisawa 252-8510, Japan.e-mail: sato.yukita@nihon-u.ac.jp

Enhancement of Glucose Toxicity by Hyperbaric Oxygen Exposure in Diabetic Rats

TOKIO MATSUNAMI,1 YUKITA SATO,1 TSUNENORI MORISHIMA,1 YOSHIHIRO MANO2 and

MASAYOSHI YUKAWA1

1Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Japan

2Department of Health Education, Division of Comprehensive Health Nursing Sciences, Graduate School of Allied Health Sciences, Tokyo Medical and Dental University, Tokyo, Japan

The side effects of hyperbaric oxygen (HBO) treatment, such as oxidative stress and oxygen toxicity, have long been of interest. However, there are no comprehensive studies evaluating such toxic effects in diabetes mellitus (DM). The purpose of this study was to determine the effects of HBO on glucose homeostasis and histological changes in pancre-atic β -cells of experimentally induced diabetic rats. A total of 24 male Wistar rats were randomly divided into 4 groups: 1) Control group, no diabetic induction without HBO treatment; 2) HBO group, exposed to 100% oxygen at 2.8 ATA (atmosphere absolute) for 2 h once daily, for 7 days; 3) DM group, diabetes induced by streptozotocin (STZ) injection; and 4) DM + HBO group, received both STZ injection and HBO exposure. HBO treat-ment, with clinically recommended pressures and duration of therapy, was started on day 5 after STZ injection, when the blood glucose levels were significantly increased. After the last HBO treatment, the pancreatic tissues were immunostained to measure the areas of insulin immunoreactive β -cells in the islets of Langerhans. The blood glucose increased significantly following exposure to HBO, with the highest levels achieved in rats, which had been treated with both HBO and diabetic induction. The area populated with insulin immunoreactive β -cells decreased significantly following diabetic induction and/or HBO exposure, with the smallest area in DM + HBO group. Thus, HBO exposure enhanced the cytotoxic effect of STZ in the β -cells of the pancreas. HBO should be cautiously employed in diabetic patients. ──── hyperbaric oxygen; streptozotocin; diabetic rat; glucose toxicity; oxidative stress.Tohoku J. Exp. Med., 2008, 216 (2), 127-132.© 2008 Tohoku University Medical Press

Hyperbaric oxygen (HBO) therapy is achieved by exposing the patient to a barometric pressure higher than ambient (1 atmosphere absolute or 1 ATA), while breathing 100% O2. Exposure to HBO leads to an increase in the amount of dissolved oxygen in the blood, result-

ing in an improvement of a variety of clinical conditions such as hypoxia, acute carbon monox-ide intoxication, air embolism and diabetic lower limb wounds (Feldmeier 2003; Lipsky et al. 2004). However, HBO administration can also lead to side effects, such as oxidative stress and

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occurred in pancreatic β -cells under HBO treat-ment. Importantly, we used pressure levels and duration of exposure to HBO typical of the clini-cal setting, in order to evaluate the toxic effects of HBO in diabetes.

MATERIALS AND METHODS

Animals and the usage policyA total of 24 adult, 10-week-old, male Wistar rats

(weight range, 280 to 320 g), bred in our laboratory, were used for the experiment. They were housed at 23 to 25°C with light from 7 :00 AM to 7 :00 PM and free access to water at all times. All animals were fed a com-mercial diet during the experiment. All study procedures were implemented in accordance with the Institutional Guidelines for Animal Experiments at the College of Bioresource Sciences, Nihon University and with the permission of the Committee of Experimental Animal in the College.

Study designThe rats (n = 24) were allowed to acclimatize for 1

week, prior to treatment. They were randomly distribut-ed into four groups, 6 rats per group, as follows: no dia-betic induction and no HBO treatment (Control); HBO treatment without diabetic induction (HBO group); dia-betic induction without HBO treatment (DM: Diabetes Mellitus group); and diabetic induction and HBO treat-ment (DM + HBO group). HBO treatment was started 5 days after the diabetic induction. The mean body weight of the animals in all groups was measured at the initial and final visits during the study period.

Induction of diabetesWistar rats were injected intraperitoneally (i.p.) with

40mg/kg streptozotocin (STZ, Wako Pure Chemical Industries, Ltd. Japan), dissolved in citrate buffer (pH 4.5), to induce diabetes as described previously (Like et al. 1976). Control and HBO rats were treated with equal volumes and concentrations of the STZ injection vehicle, citrate buffer. The blood glucose levels in the auricular veins were measured 5, 8 and 12 days after the STZ in-jection, by an automated glucose measurement equip-ment, G meter® (Sanofi-Aventis K.K. Japan). Diabetes was considered to have been induced when the glucose level reached at least 280 mg/dl (Matkovics et al. 1997).

HBO exposureHBO exposure was set at a pressure of 2.8 ATA for

oxygen toxicity (Harabin et al. 1990; Chavko et al. 1996; Speit et al. 2002). Reported side effects have often been associated with exposure to levels of HBO much higher than those generally used for treating clinical conditions. The clinical-ly approved maximum pressure and duration of HBO exposure are 3 ATA and 120 min, respec-tively (Feldmeier 2003), although the most com-monly-used protocol for standard therapeutic purposes is slightly lower (1.8 to 2.8 ATA for 60 to 90 min) (Benedetti et al. 2004). The oxidative effects of HBO have been investigated in animals and humans (Oter et al. 2005; Eken et al. 2005). One study on the effects of HBO in rats revealed that, following 2 h of HBO exposure at 3 ATA, elevated levels of oxidative stress markers, thio-barbituric acid reactive substances (TBARS) and superoxide dismutase (SOD) were found in the lung, brain and erythrocytes (Oter et al. 2005). However, these authors used pressure conditions that were higher than the standard clinical treat-ment. Therefore, it appears that adverse side effects of HBO exposure have occurred under conditions of overdose, at least as far as the stan-dard clinical levels of exposure are concerned. Therefore, in order to evaluate the safety of HBO exposure, it is necessary to use conditions (in terms of pressure and duration) similar to those used in the clinical setting.

Although HBO has been used to treat several medical conditions, its potential side effects should not be ignored. As mentioned above, HBO therapy has been used to facilitate the repair of diabetic lower limb wounds (Tibbles et al. 1996). However, diabetes is a condition that induces oxidative stress (Oberley 1996; Brownlee et al. 2001), and further exposure to HBO may only serve to exacerbate this stress, and thereby accelerate the progress of the illness. The side effects of HBO therapy in diabetic patients have been poorly investigated. Moreover, there is an insufficient number of adequate models available with which to evaluate the toxic effects of HBO associated with this stress. In this study, using an animal model of diabetes, we examined the physi-ological parameters associated with glucose homeostasis and the histological changes that

Enhancement of Glucose Toxicity by HBO 129

2 h once daily, for 7 days for all experiments; this proto-col reflects that used in the clinical setting. A hyperbaric chamber for small animals (Nakamura Tekko-sho K.K., Tokyo, Japan) was used for HBO exposure. The ventila-tion rate was 4-5 L/min. All administrations were started at the same hour in the morning (10 AM) to exclude any confounding issues associated with biological rhythm changes.

Tissue preparationAfter the final exposure to HBO, the animals were

weighed and anesthetized with sodium pentobarbital 40 mg/kg i.p. Pancreatic tissues were harvested from the sacrificed animals, and the fragments from tissues were fixed in 10% neutral formalin solution, embedded in par-affin and then stained with immunostaining for insulin, according to the method of Coskun et al. (2005) for his-topathological investigations.

Image analysisThe areas of insulin immunoreactive β -cells in each

group of rats were compared using images obtained using the Image J software version 1.8 system (Wayne Rasband National Institutes of Health, USA). Forty Langerhans islets were chosen at random from each group of individual rats. The percentage of insulin immunoreactive β -cells was calculated for each group.

Statistical analysisThe collected and calculated data were expressed as

mean ± standard deviation (S.D.). Student’s t-test was used to determine significant differences between the control and hyperbaric groups. For the analysis of the immunohistochemical data, a nonparametric test (Kruskal-Wallis) was used. Differences were considered

statistically significant if p < 0.05.

RESULTS

The baseline body weight of the rats at beginning of the study was similar and in all groups. At the end of the treatment (after 12 days), there was no difference in body weight between control and HBO rats (mean group weights ranged from 307 to 313 g). However dia-betic rats presented with weight loss, and the body weight of DM + HBO rats (262 ± 10 g; mean ± S.D.), in particular, decreased significantly (P < 0.01), when compared with the rats in the other 3 groups (control, 313 ± 5 g; HBO, 307 ± 4 g; DM, 280 ± 9 g).

Biochemical findingsThe blood glucose levels of the DM + HBO

group were significantly higher (P < 0.05) 8 and 12 days after diabetes induction, compared with the DM group (Fig. 1). No significant differences were observed between any other groups or experimental periods.

Histological findingsThe pancreatic islet cells were histologically

normal in the control and HBO groups. Strongly stained, anti-insulin positive cells were observed in the islets of the pancreatic tissues from the non-diabetic rats in these control and HBO groups (Fig. 2). In contrast, in the DM and the DM + HBO groups, insulin antigen-positive signals were weak in the majority of the islets (Fig. 3). Insulin

Fig. 1. Glucose levels in non-diabetic Wistar (WR) and diabetic WR rats. Values are expressed as the mean ± standard deviation (n = 6).

　　*P < 0.01, **P < 0.001 compared with control value.

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immunoreactive β -cells were measured in the islets of Langerhans. The percentage of insulin immunoreactive β -cells is shown in Table 1. The area of insulin immunoreactive β -cells in the DM and DM + HBO groups decreased significantly (P < 0.001), when compared with the HBO group.

Furthermore, HBO treatment was associated with a significant decrease in insulin immunoreactive β -cells (P < 0.001; DM + HBO vs. DM).

DISCUSSION

HBO therapy is a unique method used in the

Fig. 2. Non-diabetic rats in the non-HBO (control) and hyperbaric (HBO) groups, showing normal cells in the islets of Langerhans and also showing β -cells in the islets of Langerhans that are strongly stained with the anti-insulin antibody. Immunoperoxidase, haematoxylin counterstain × 320. Scale bar = 50 μm.

Fig. 3. Diabetic rats in the non-HBO (DM) and hyperbaric (DM + HBO) groups, showing β -cells in the islets of Langerhans with weak insulin-immunoreactivity in the majority of β -cells. Immunoperoxi-dase, haematoxylin counterstain × 320. Scale bar = 50 μm.

TABLE 1. Comparison of the areas with the insulin immunoreactive β -cells in the islets of Langerhans.

Group Mean area of insulin immunoreactive β -cells in islets

Control 81.32 ± 6.66%HBO 80.91 ± 8.65%DM 18.33 ± 3.29%*DM + HBO 8.01 ± 3.31%**

Non-diabetic, non-HBO rats (control) and hyperbaric (HBO) groups; diabetic, non-HBO rats (DM) and hyperbaric (DM + HBO) groups.

Kruskal-Wallis test was used for statistical analysis. Values are expressed as mean ± S.D., and n = 6 animals for all groups.

*P < 0.001; DM vs. HBO. **P < 0.001; DM + HBO vs. DM.Note that HBO treatment was associated with a significant decrease in insulin

immunoreactive β -cells in DM rats.

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treatment of a variety of illnesses and conditions, such as carbon monoxide poisoning, decompres-sion sickness, osteomyelitis, diabetic foot, and impaired wound healing (Feldmeier 2003). The Undersea and Hyperbaric Medical Society (UHMS) has an approved list of clinical condi-tions where HBO treatment is indicated, includ-ing, amongst others, the above mentioned disor-ders (Feldmeier 2003). Nevertheless, this should not be considered as exhaustive, as there is an abundance of reports which suggest many suc-cessful cures obtained with HBO treatment for conditions other than those on the UHMS list. Although HBO treatment seems to be a ‘cure-all’ therapy, nevertheless, potential and known side effects should be studied further to avoid the excessive use of HBO in the clinical setting.

The causes of the observed side effects fall into two categories: firstly, those due to the poten-tial risk of oxygen toxicity itself during the treat-ment period; secondly, those due to the use of a pressure and duration of HBO administration that exceeds the approved therapeutic limits (currently set at, a maximum 3 ATA pressure and 2 h dura-tion). Oxygen toxicity, manifested for example by a slight increase in SOD and reduced glutathi-one peroxidase (GPx) and catalase (CAT) activity, has been reported in both the lung and brain of rats and guinea pigs, in situations where although the HBO exposure pressures were clinically acceptable, the duration of the session was rather longer than the recommended 2 hours (Harabin et al. 1990). Direct toxic effects of HBO have been observed under conditions with relatively higher pressures and longer durations, such as 4 to 5 ATA and more than 2 hours, respectively (Harabin et al. 1990). In the present study, we used an HBO exposure protocol which followed clinically used pressures and duration, and investigated the levels of blood glucose, and histological changes in the pancreatic β -cells of STZ-induced diabetic rats, a recognized model of type 1 diabetes mellitus (Kakkar et al. 1995). Our results showed that glucose levels were not improved in HBO-treated diabetic rats, suggesting that HBO administration might have the ability to enhance the functional disorder of pancreatic islet cells. A recent study

has reported that HBO exposure at a pressure of 2.8 ATA for 2 h once daily for 7 days elevated hemorheological data and the TBARS levels of erythrocyte membranes in diabetic rats (Liu et al. 2003). These authors showed significant changes of the hemorheological parameters in diabetic rats associated with the administration of HBO, how-ever, they did not study the duration of the chang-es of blood glucose levels or perform any histo-logical analysis. We have shown for the first time in STZ-diabetic rats, that the rise in blood glucose levels started after the beginning of HBO treat-ment, administrated at pressure levels and for a duration of time used in the clinical setting. Furthermore, this present study also showed that the insulin-producing β -cells areas of diabetic rats receiving HBO treatment were significantly reduced compared with those of the diabetic con-trol group (i.e., no HBO treatment). STZ, a mono-functional nitrosourea derivative, is one of the most commonly used substances to induce diabetes in experimental animals (Evans et al. 1965; Szkudelski 2001). Previous evidence sug-gests that STZ may damage pancreatic β -cells and could induce oxidative stress (Ohkuwa et al. 1995).

On the other hand, it has been shown that blood glucose levels were decreased by HBO treatment in GK rats, an animal model of spontaneous type 2 diabetes, suggesting that HBO could interfere with the activity of the natural anti-oxidative defense system and thus promote de novo generation of free radicals (Ihara et al. 1999; Maritim et al. 2003). Our results suggest that the mode of action of HBO against the glu-cose control system might be different in STZ- and GK-induced diabetic rats. The possible side effects of HBO seen in this study, namely the elevation of glucose levels in the peripheral blood and the damage to β -cells, might occur as a result of the enhancement of STZ cytotoxicity by HBO administration. One factor behind the enhance-ment of glucose toxicity might be oxidative stress, as described previously (Ihara et al. 1999). Our experimental system, which uses STZ-induced diabetes plus HBO treatment, could be used as a model for evaluating oxidative stress, through the

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monitoring of unusual increases in glucose levels. Nevertheless, in cases where cytotoxicity occurs during HBO administration, due to factors other than STZ, it will also be necessary to investigate the influence of HBO. For the safe employment of HBO for therapeutic purpose, it will be impor-tant to fully understand the nature of the toxic effects of HBO with respect to oxidative stress.

AcknowledgmentsThis study was partially supported by the

Academic Frontier Project “Surveillance and control for zoonoses” from Ministry of Education, Culture, Sports, Science and Technology, Japan.

ReferencesBenedetti, S., Lamorgese, A., Piersantelli, M., Pagliarani, S.,

Benvenuti, F. & Canestrari, F. (2004) Oxidative stress and antioxidant status in patients undergoing prolonged expo-sure to hyperbaric oxygen. Clin. Biochem., 37, 312-317.

Brownlee, M. (2001) Biochemistry and molecular cell biology of diabetic complications. Nature, 414, 813-820.

Chavko, M. & Harabin, A.L. (1996) Regional lipid peroxida-tion and protein oxidation in rat brain after hyperbaric oxy-gen exposure. Free Radic. Biol. Med., 20, 973-978.

Coskun, O., Kanter, M., Korkmaz, A. & Oter, S. (2005) Quer-cetin, a flavonoid antioxidant, prevents and protects strep-tozotocin-induced oxidative stress and beta-cell damage in rat pancreas. Pharmacol. Res., 51, 117-123.

Eken, A., Aydin, A., Sayal, A., Ustündağ, A., Duydu, Y. & Dündar, K. (2005) The effects of hyperbaric oxygen treat-ment on oxidative stress and SCE frequencies in humans. Clin. Biochem., 38, 1133-1137.

Evans, J.S., Gerritsen, G.C., Mann, K.M. & Owen, S.P. (1965) Antitumor and hyperglycemic activity of streptozotocin (NSC-37917) and its cofactor, U-15,774. Cancer Chemother. Rep., 48, 1-6.

Feldmeier, J.J. (2003) Hyperbaric oxygen: indications and results; the hyperbaric oxygen therapy committee report. Undersea. Hyperb. Med. Soc.

Harabin, A.L., Braisted, J.C. & Flynn, E.T. (1990) Response of

antioxidant enzymes to intermittent and continuous hyper-baric oxygen. J. Appl. Physiol., 69, 328-335.

Ihara, Y., Toyokuni, S., Uchida, K., Odaka, H., Tanaka, T., Ikeda, H., Hiai, H., Seino, Y. & Yamada, Y. (1999) Hyper-glycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes, 48, 927-932.

Kakkar, R., Kalra, J., Mantha, S.V. & Prasad, K. (1995) Lipid peroxidation and activity of antioxidant enzymes in diabet-ic rats. Mol. Cell. Biochem., 151, 113-119.

Like, A.A. & Rossini, A.A. (1976) Streptozotocin-induced pan-creatic insulitis: new model of diabetes mellitus. Science., 193, 415-417.

Lipsky, B.A., Berendt, A.R., Deery, H.G., Embil, J.M., Joseph, W.S., Karchmer, A.W., LeFrock, J.L., Lew, D.P., Mader, J.T., Norden, C. & Tan, J.S. (2004) Diagnosis and treat-ment of diabetic foot infections. Clin. Infect. Dis., 39, 885-910.

Liu, D.Z., Chien, S.C., Tseng, L.P. & Yang, C.B. (2003) The influence of hyperbaric oxygen on hemorheological param-eters in diabetic rats. Biorheol., 40, 605-612.

Maritim, A.C., Sanders, R.A. & Watkins, J.B. 3rd. (2003) Effects of alpha-lipoic acid on biomarkers of oxidative stress in streptozotocin-induced diabetic rats. J. Nutr. Biochem., 14, 288-294.

Matkovics, B., Kotorman, M., Varga, I.S., Hai, D.Q. & Varga, C. (1997) Oxidative stress in experimental diabetes induced by streptozotocin. Acta Physiol. Hung., 85, 29-38.

Oberley, L.W. (1996) Free radicals and diabetes. Free Radic. Biol. Med., 5, 113-124.

Ohkuwa, T., Sato, Y. & Naoi, M. (1995) Hydroxyl radical for-mation in diabetic rats induced by streptozotocin. Life Sci., 56, 1789-1798.

Oter, S., Korkmaz, A., Topal, T., Ozcan, O., Sadir, S., Ozler, M., Ogur, R. & Bilgic, H. (2005) Correlation between hyper-baric oxygen exposure pressures and oxidative parameters in rat lung, brain, and erythrocytes. Clin. Biochem., 38, 706-711.

Speit, G., Dennog, C., Radermacher, P. & Rothfuss, A. (2002) Genotoxicity of hyperbaric oxygen. Mutat. Res., 512, 111-119.

Szkudelski, T. (2001) The mechanism of alloxan and strepto-zotocin action in β cell of the rat pancreas. Physiol. Res., 50, 537-546.

Tibbles, P.M. & Edelsberg, J.S. (1996) Hyperbaric-oxygen therapy. N. Engl. J. Med., 334, 1642-1648.