About this Journal Submit a Manuscript Table of Contents
Oxidative Medicine and Cellular Longevity
Volume 2012 (2012), Article ID 381763, 5 pages
http://dx.doi.org/10.1155/2012/381763
Research Article

Oxygen Concentration-Dependent Oxidative Stress Levels in Rats

1Laboratory of Cell Biology and Life Science, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan
2Department of Rehabilitation Science, Kobe University Graduate School of Health Sciences, Kobe 654-0142, Japan
3Department of Food Sciences and Nutrition, Nagoya Women's University, Nagoya 467-8610, Japan

Received 19 April 2012; Revised 5 August 2012; Accepted 8 August 2012

Academic Editor: Jose Luis Martin-Ventura

Copyright © 2012 Fumiko Nagatomo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction. We determined derivatives of reactive oxygen metabolites (dROMs) as an index of oxidative stress level (oxidant capacity) and biochemical antioxidant potential (BAP) as an index of antioxidant capacity in rats exposed to different oxygen concentrations. Methods. Male Wistar rats were exposed to 14.4%, 20.9%, 35.5%, 39.8%, 62.5%, and 82.2% oxygen at 1 atmosphere absolute for 24 h. Serum levels of dROMs and BAP were examined by using a free radical and antioxidant potential determination device. The morphological characteristics of red blood cells were examined by phase contrast microscopy. Results. There were no differences in the levels of dROMs in rats exposed to 14.4%, 20.9%, and 35.5% oxygen. However, the levels of dROMs increased in the rats exposed to 39.8% and 62.5% oxygen. The levels of dROMs were the highest in the rats exposed to 82.2% oxygen. There were no differences in the levels of BAP with respect to the oxygen concentration. Morphological changes in the red blood cells induced by oxidative attack from reactive oxygen species were observed in the rats exposed to 39.8%, 62.5%, and 82.2% oxygen. Conclusion. Our results suggest that exposure to oxygen concentrations higher than 40% for 24 h induces excessive levels of oxidative stress in rats.

1. Introduction

Supplemental oxygen is used in treatment and as a countermeasure for acute and chronic diseases. When pilots of unpressurized aircrafts fly to areas at high altitudes, when climbers ascend high-altitude peaks and outpace their ability to acclimatize, or when divers inhaling compressed air return to the surface, the external pressure on the body decreases and the dissolved inert gases come out of solution in the form of bubbles in the body on depressurization [1, 2]. The resulting decompression sicknesses and air embolisms are initially treated by inhalation of oxygen-enriched air or exposure to mild hyperbaric oxygen at 1.25 atmospheres absolute (ATA) until hyperbaric oxygen therapy (100% oxygen delivered at 2-3 ATA) is administered [36]. Hypoxic or breathless patients with chronically obstructive pulmonary disease (COPD), who have low levels of oxygen in their blood, require oxygen at concentrations greater than that in room air to achieve arterial oxygen saturations between 88% and 92% [7].

Oxygen therapy with or without pressure is associated with the risk of oxygen toxicity and excessive oxidative stress. Oxidative stress plays as key role in the pathogenesis of many diseases and their complications; the generation of free radicals and increased levels of oxidative stress are associated with atherosclerosis, cataract, retinopathy, myocardial infarction, hypertension, diabetes, renal failure, and uremia [810]. However, there are no data available on changes in the oxidative stress level and antioxidant capacity after exposure to different concentrations of oxygen. The analytical measurement of oxidative stress markers has been difficult because of the short half-life and high reactivity of the majority of reactive oxygen species and the applicability of measurement methods [11]. Blood samples are the appropriate biological materials for assessing the status of oxidants and antioxidants. A unique system for the evaluation of oxidative stress levels and antioxidant capacity in the blood has been developed [12]. This evaluation approach is based on the free radical analytical system that mainly analyzes lipid hydroperoxides, which are relatively stable in the blood. This system has been used for both animal and human sera, which confirms its applicability [1315].

In this study, we examined the derivatives of reactive oxygen metabolites (dROMs) as an index of oxidative stress levels (oxidant capacity) and the biochemical antioxidant potential (BAP) as an index of antioxidant capacity in rats exposed to different concentrations of oxygen at 1 ATA for 24 h.

2. Materials and Methods

2.1. Experimental Animals

All experimental and animal care procedures were performed in accordance with the guidelines stated in the Guide for the Care and Use of Laboratory Animals issued by the Institutional Animal Experiment Committee of Kyoto University (Kyoto, Japan).

2.2. Exposure to Different Concentrations of Oxygen

Ten-week-old male Wistar rats weighing between 200 g and 226 g were divided into 6 groups ( 𝑛 = 5 for each group). The individual groups were exposed to air containing low or high concentration of oxygen in a chamber (75 × 130 × 85 cm) at 1 ATA for 24 h by using a low-oxygen inhaler (Terucom Corp., Yokohama, Japan) or an oxygen concentrator (Ikiken Corp., Sayama, Japan), respectively. When the air chamber contained less or more than 20.9% oxygen, air with 2 different concentrations of oxygen from 2 tubes was transported to the aspirator: one tube contained normal air (20.9% oxygen) and the other tube had air containing 13% or 84% oxygen, which was procured from the low-oxygen inhaler or the oxygen concentrator, respectively. Then, the aspirator pumped mixed air into the chamber at the rate of 1 L/min. The low or high oxygen concentration in the chamber was adjusted by separately regulating the air flow from these 2 tubes. Normal air was transported by only 1 tube, while the air containing 20.9% oxygen was retained in the chamber. The oxygen concentration in the chamber was determined by using an oxygen monitor (Max O2+AE; Maxtec Inc., UT, USA) attached to the chamber. The rats were maintained in individual, uniformly sized standard cages (30 × 40 × 20 cm) in the chamber. The room was maintained at 2 2 ± 2 °C with 45%–55% relative humidity. Food and water were provided ad libitum.

2.3. Measurements of dROMs and BAP

The levels of dROMs and BAP were determined after the rats were exposed to different concentrations of oxygen. Blood samples were obtained from the tail of fully conscious rats and evaluated photometrically. A free radical and antioxidant potential determination device (Free Radical Analytical System 4; Health & Diagnostics, Grosseto, Italy) was used to automatically measure the levels of dROMs and BAP.

The dROMs are used as an index to determine the level of oxidative stress (oxidant capacity) by measuring the amount of organic hydroperoxide (ROOH) converted into radicals that oxidize N,N-diethyl-p-phenylenediamine [12, 16]. The levels of dROMs were expressed in Carr units (1 U Carr corresponds to 0.08 mg hydroperoxide/100 mL H2O2). The BAP is used as an index to determine the biological antioxidant capacity and is measured on the basis of the capacity of the plasma sample to reduce ferric ions to ferrous ions. After blood samples were obtained, the rats were killed by intraperitoneal overdose of sodium pentobarbital.

2.4. Red Blood Cell Morphology

Using blood samples, the morphological profiles of red blood cells were observed by phase contrast microscopy (Nikon 80iF-PH-15; Tokyo, Japan).

2.5. Statistics

Means and standard deviations were calculated from the individual values by using standard procedures. One-way analysis of variance (ANOVA) was used to evaluate the mean differences among the 6 groups. When ANOVA analyses revealed significant differences in mean values, the groups were further compared using Scheffé’s post hoc tests. A probability level of 0.05 was considered significant.

3. Results

The levels of dROMs in rats exposed to 39.8% and 62.5% oxygen were higher than those in the rats exposed to 14.4%, 20.9%, and 35.5% oxygen (Figure 1(a)). The level of dROMs in the rats exposed to 82.2% oxygen was the highest among the 6 groups. There were no differences in the levels of BAP among the 6 groups (Figure 1(b)). Morphological changes in red blood cells were observed in the rats exposed to 39.8%, 62.5%, and 82.2% oxygen (Figure 2).

fig1
Figure 1: Levels of derivatives of reactive oxygen metabolites (a) and biochemical antioxidant potential (b) of rats after exposure to different concentrations of oxygen for 24 h. Values are expressed as the mean and standard deviation ( 𝑛 = 5 for each group). Five dots on the right side of the bar are the individual values of rats in each group. dROMs: derivatives of reactive oxygen metabolites; BAP: biochemical antioxidant potential. * 𝑃 < 0 . 0 5 compared with values of 14.4%, 20.9%, and 35.5% oxygen; ** 𝑃 < 0 . 0 5 compared with values of 14.4%, 20.9%, 35.5%, 39.8%, and 62.5% oxygen.
fig2
Figure 2: Morphological features of red blood cells after 24 h of exposure to different concentrations of oxygen: (a) 14.4%, (b) 20.9%, (c) 35.5%, (d) 39.8%, (e) 62.5%, and (f) 82.2%. RBC: red blood cell; WBC: white blood cell. Scale bar is 10 μm.

4. Discussion

4.1. Exposure to Low Concentration of Oxygen

Acclimatization at high altitude results in changes in the respiratory, cardiovascular, and hematologic systems, which enhance oxygen delivery to the cells and tissues [17]. Decompression sickness occurs between initial hypoxic conditions and the onset of acclimatization, and the incidence and severity of the sickness depend on the rate of ascent, the altitude attained, and physiological susceptibility of individuals [5].

There is little data available regarding the oxidative stress level under low concentrations of oxygen. In this study, we examined the oxidative stress levels in rats exposed to low concentrations of oxygen. In rats exposed to 14.4% oxygen for 24 h, which is equivalent to the oxygen concentration at 3500 m/11500 feet altitude, no change was observed in the oxidative stress level (Figure 1(a)). Therefore, we conclude that low concentrations of oxygen do not induce excessive oxidative stress.

4.2. Exposure to High Concentration of Oxygen

Hyperbaric oxygen at 2-3 ATA with 100% oxygen induces acute changes, such as increased blood pressure and further reduction in heart rate [18], and causes chronic diseases like cataract formation [1922]; hyperbaric oxygen is generally safe when pressures do not exceed 3 ATA and the length of treatment is less than 120 min [23, 24]. However, hyperbaric oxygen has been reported to increase the levels of reactive oxygen species [2527].

Patients with COPD inhale high concentrations of oxygen, generally up to 50% oxygen (fraction of inspired oxygen, FIO2) when the partial pressure of oxygen in the arterial blood (PaO2) is below 55 mmHg; these patients occasionally exhibit voluntary respiration failure, consciousness disturbance, and atelectasis when inhaling high concentrations of oxygen.

Oxygen therapy with or without pressure might induce excessive levels of oxidative stress. Excessive levels of oxidative stress are associated with many diseases, including atherosclerosis, cataract, retinopathy, myocardial infarction, hypertension, renal failure, and uremia [810]. In this study, we examined the oxidative stress levels in rats exposed to high concentrations of oxygen.

No change in the oxidative stress level was observed at 35.5% oxygen for 24 h (Figure 1(a)). We previously examined the effects of mild hyperbaric oxygen at 1.25 ATA with 36% oxygen on the neuromuscular system, including spinal motoneurons and their innervating muscle fibers [28, 29], type 2 diabetes [3033], hypertension [34], type II collagen-induced arthritis [35], age-related decline in muscle oxidative capacity [36], and diabetes-induced cataracts [37] in mice and rats. Therefore, the data observed in this study suggest that mild hyperbaric oxygen at 1.25 ATA with 36% oxygen is effective for the inhibition and improvement of many metabolic diseases [2837], without producing excessive levels of oxidative stress.

In contrast, 39.8% and 62.5% oxygen for 24 h induced excessive levels of oxidative stress, and the level of dROMs was the highest at 82.2% oxygen (Figure 1(a)). Oxidative stress level increases when the production of reactive oxygen species is markedly greater than the intrinsic antioxidant defenses. Patients with COPD inhale high concentrations of oxygen, and the possibility of accumulating excessive levels of oxidative stress will be greater when the inhaled oxygen concentration (FIO2) is high. Therefore, we conclude that exposure to 40% oxygen for 24 h is a threshold for inducing an excessive level of oxidative stress.

In our previous study [15], we observed that obese rats with metabolic syndrome accompanied by insulin resistance, impaired glucose metabolism, and dyslipidemia had lower levels of BAP than normal Wistar rats. In addition, we observed that mild hyperbaric exposure at 1.25 ATA with 36% oxygen improves the levels of BAP in rats with hypertension [34]. These studies [15, 34] suggest that changes in the levels of BAP are reflected as an index of antioxidant capacity. In this study, we expected that exposure to higher concentrations of oxygen would decrease the levels of BAP compared with exposure to 20.9% oxygen because the levels of dROMs were increased by exposure to 39.8%, 62.5%, and 82.2% oxygen (Figure 1(a)). However, there was no difference in the levels of BAP among different concentrations of oxygen (Figure 1(b)). These results suggest that the antioxidant capacity is not affected by both low and high concentrations of oxygen, and thus, the antioxidant capacity did not change, at least after 24 h. However, we did not examine antioxidant enzyme levels of rats exposed to different concentrations of oxygen. In our subsequent study, we plan to examine the levels of BAP and antioxidant enzymes, for example, superoxide dismutase, catalase, and glutathione peroxidase, in rats exposed to different concentrations of oxygen for more than 24 h.

Transformed red blood cells, which were induced by oxidative attack from reactive oxygen species, were observed by exposure to 39.8%, 62.5%, and 82.2% oxygen (Figure 2); these findings are consistent with the increased levels of dROMs at these oxygen concentrations (Figure 1(a)). Therefore, we conclude that morphological changes in the red blood cells are linked to increased levels of oxidative stress.

5. Conclusion

Exposure to oxygen concentrations higher than 40% for 24 h induces excessive levels of oxidative stress.

Conflict of Interests

The authors declare that they have no conflict of interests.

References

  1. E. C. Parker, S. S. Survanshi, P. B. Massell, and P. K. Weathersby, “Probabilistic models of the role of oxygen in human decompression sickness,” Journal of Applied Physiology, vol. 84, no. 3, pp. 1096–1102, 1998. View at Scopus
  2. A. J. F. Macmillan, “Chapter 3. Sub-atmospheric decompression sickness,” in Aviation Medicine, pp. 19–25, Butterworth Heinemann, Oxford, UK, 3rd edition, 1999.
  3. J. F. Kasic, H. M. Smith, and R. I. Gamow, “A self-contained life support system designed for use with a portable hyperbaric chamber,” Biomedical Sciences Instrumentation, vol. 25, pp. 79–81, 1989. View at Scopus
  4. J. F. Kasic, M. Yaron, R. A. Nicholas, J. A. Lickteig, and R. Roach, “Treatment of acute mountain sickness: hyperbaric versus oxygen therapy,” Annals of Emergency Medicine, vol. 20, no. 10, pp. 1109–1112, 1991. View at Publisher · View at Google Scholar · View at Scopus
  5. C. Imray, A. Wright, A. Subudhi, and R. Roach, “Acute mountain sickness: pathophysiology, prevention, and treatment,” Progress in Cardiovascular Diseases, vol. 52, no. 6, pp. 467–484, 2010. View at Publisher · View at Google Scholar · View at Scopus
  6. G. J. Butler, N. Al-Waili, D. V. Passano et al., “Altitude mountain sickness among tourist populations: a review and pathophysiology supporting management with hyperbaric oxygen,” Journal of Medical Engineering & Technology, vol. 35, no. 3-4, pp. 197–207, 2011. View at Publisher · View at Google Scholar · View at Scopus
  7. M. A. Austin, K. E. Wills, L. Blizzard, E. H. Walters, and R. Wood-Baker, “Effect of high flow oxygen on mortality in chronic obstructive pulmonary disease patients in prehospital setting: randomised controlled trial,” British Medical Journal, vol. 341, p. c5462, 2010. View at Publisher · View at Google Scholar · View at Scopus
  8. C. M. Maier and P. H. Chan, “Role of superoxide dismutases in oxidative damage and neurodegenerative disorders,” Neuroscientist, vol. 8, no. 4, pp. 323–334, 2002. View at Scopus
  9. K. K. Griendling and G. A. FitzGerald, “Oxidative stress and cardiovascular injury. Part I: basic mechanisms and in vivo monitoring of ROS,” Circulation, vol. 108, no. 16, pp. 1912–1916, 2003. View at Publisher · View at Google Scholar · View at Scopus
  10. I. Dalle-Donne, R. Rossi, R. Colombo, D. Giustarini, and A. Milzani, “Biomarkers of oxidative damage in human disease,” Clinical Chemistry, vol. 52, no. 4, pp. 601–623, 2006. View at Publisher · View at Google Scholar · View at Scopus
  11. D. Pitocco, F. Zaccardi, E. Di Stasio et al., “Oxidative stress, nitric oxide, and diabetes,” Review of Diabetic Studies, vol. 7, no. 1, pp. 15–25, 2010. View at Publisher · View at Google Scholar · View at Scopus
  12. A. Alberti, L. Bolognini, D. Macciantelli, and M. Caratelli, “The radical cation of N,N-diethyl-para-phenylendiamine: a possible indicator of oxidative stress in biological samples,” Research on Chemical Intermediates, vol. 26, no. 3, pp. 253–267, 2000. View at Scopus
  13. C. Vassalle, C. Boni, P. Di Cecco, R. Ndreu, and G. C. Zucchelli, “Automation and validation of a fast method for the assessment of in vivo oxidative stress levels,” Clinical Chemistry and Laboratory Medicine, vol. 44, no. 11, pp. 1372–1375, 2006. View at Publisher · View at Google Scholar · View at Scopus
  14. A. Pasquini, E. Luchetti, V. Marchetti, G. Cardini, and E. L. Iorio, “Analytical performances of d-ROMs test and BAP test in canine plasma. Definition of the normal range in healthy Labrador dogs,” Veterinary Research Communications, vol. 32, no. 2, pp. 137–143, 2008. View at Publisher · View at Google Scholar · View at Scopus
  15. F. Nagatomo, N. Gu, H. Fujino, I. Takeda, K. Tsuda, and A. Ishihara, “Skeletal muscle characteristics of rats with obesity, diabetes, hypertension, and hyperlipidemia,” Journal of Atherosclerosis and Thrombosis, vol. 16, no. 5, pp. 576–585, 2009. View at Scopus
  16. R. Trotti, M. Carratelli, and M. Barbieri, “Performance and clinical application of a new, fast method for the detection of hydroperoxides in serum,” Panminerva Medica, vol. 44, no. 1, pp. 37–40, 2002. View at Scopus
  17. S. J. Paralikar and J. H. Paralikar, “High-altitude medicine,” Indian Journal of Occupational and Environmental Medicine, vol. 14, no. 1, pp. 6–12, 2010. View at Publisher · View at Google Scholar · View at Scopus
  18. N. S. Al-Waili, G. J. Butler, J. Beale et al., “Influences of hyperbaric oxygen on blood pressure, heart rate and blood glucose levels in patients with diabetes mellitus and hypertension,” Archives of Medical Research, vol. 37, no. 8, pp. 991–997, 2006. View at Publisher · View at Google Scholar · View at Scopus
  19. B. M. Palmquist, B. Philipson, and P. O. Barr, “Nuclear cataract and myopia during hyperbaric oxygen therapy,” British Journal of Ophthalmology, vol. 68, no. 2, pp. 113–117, 1984. View at Scopus
  20. F. J. Giblin, V. A. Padgaonkar, V. R. Leverenz et al., “Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen,” Experimental Eye Research, vol. 60, no. 3, pp. 219–235, 1995. View at Publisher · View at Google Scholar · View at Scopus
  21. V. A. Padgaonkar, V. R. Leverenz, K. E. Fowler, V. N. Reddy, and F. J. Giblin, “The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper,” Experimental Eye Research, vol. 71, no. 4, pp. 371–383, 2000. View at Publisher · View at Google Scholar · View at Scopus
  22. L. B. Gesell and A. Trott, “De novo cataract development following a standard course of hyperbaric oxygen therapy,” Undersea and Hyperbaric Medicine, vol. 34, no. 6, pp. 389–392, 2007. View at Scopus
  23. P. M. Tibbles and J. S. Edelsberg, “Hyperbaric-oxygen therapy,” New England Journal of Medicine, vol. 334, no. 25, pp. 1642–1648, 1996. View at Scopus
  24. R. M. Leach, P. J. Rees, and P. Wilmshurst, “ABC of oxygen: hyperbaric oxygen therapy,” British Medical Journal, vol. 317, no. 7166, pp. 1140–1143, 1998. View at Scopus
  25. C. K. Narkowicz, J. H. Vial, and P. W. McCartney, “Hyperbaric oxygen therapy increases free radical levels in the blood of humans,” Free Radical Research Communications, vol. 19, no. 2, pp. 71–80, 1993. View at Scopus
  26. S. Öter, A. Korkmaz, T. Topal et al., “Correlation between hyperbaric oxygen exposure pressures and oxidative parameters in rat lung, brain, and erythrocytes,” Clinical Biochemistry, vol. 38, no. 8, pp. 706–711, 2005. View at Publisher · View at Google Scholar · View at Scopus
  27. S. Öter, T. Topal, S. Sadir et al., “Oxidative stress levels in rats following exposure to oxygen at 3 atm for 0-120 min,” Aviation, Space, and Environmental Medicine, vol. 78, no. 12, pp. 1108–1113, 2007. View at Publisher · View at Google Scholar · View at Scopus
  28. A. Ishihara, F. Kawano, T. Okiura, F. Morimatsu, and Y. Ohira, “Hyperbaric exposure with high oxygen concentration enhances oxidative capacity of neuromuscular units,” Neuroscience Research, vol. 52, no. 2, pp. 146–152, 2005. View at Publisher · View at Google Scholar · View at Scopus
  29. A. Matsumoto, T. Okiura, F. Morimatsu, Y. Ohira, and A. Ishihara, “Effects of hyperbaric exposure with high oxygen concentration on the physical activity of developing rats,” Developmental Neuroscience, vol. 29, no. 6, pp. 452–459, 2007. View at Publisher · View at Google Scholar · View at Scopus
  30. K. Yasuda, N. Aoki, T. Adachi et al., “Hyperbaric exposure with high oxygen concentration inhibits growth-associated increase in the glucose level of diabetic Goto-Kakizaki rats,” Diabetes, Obesity and Metabolism, vol. 8, no. 6, pp. 714–715, 2006. View at Publisher · View at Google Scholar · View at Scopus
  31. K. Yasuda, T. Adachi, N. Gu et al., “Effects of hyperbaric exposure with high oxygen concentration on glucose and insulin levels and skeletal muscle-fiber properties in diabetic rats,” Muscle & Nerve, vol. 35, no. 3, pp. 337–343, 2007. View at Publisher · View at Google Scholar · View at Scopus
  32. A. Matsumoto, F. Nagatomo, K. Yasuda, K. Tsuda, and A. Ishihara, “Hyperbaric exposure with high oxygen concentration improves altered fiber types in the plantaris muscle of diabetic Goto-Kakizaki rats,” Journal of Physiological Sciences, vol. 57, no. 2, pp. 133–136, 2007. View at Publisher · View at Google Scholar · View at Scopus
  33. N. Gu, F. Nagatomo, H. Fujino, I. Takeda, K. Tsuda, and A. Ishihara, “Hyperbaric oxygen exposure improves blood glucose level and muscle oxidative capacity in rats with type 2 diabetes,” Diabetes Technology & Therapeutics, vol. 12, no. 2, pp. 125–133, 2010. View at Publisher · View at Google Scholar · View at Scopus
  34. F. Nagatomo, H. Fujino, I. Takeda, and A. Ishihara, “Effects of hyperbaric oxygenation on blood pressure levels of spontaneously hypertensive rats,” Clinical and Experimental Hypertension, vol. 32, no. 3, pp. 193–197, 2010. View at Publisher · View at Google Scholar · View at Scopus
  35. F. Nagatomo, N. Gu, H. Fujino et al., “Effects of exposure to hyperbaric oxygen on oxidative stress in rats with type II collagen-induced arthritis,” Clinical and Experimental Medicine, vol. 10, no. 1, pp. 7–13, 2010. View at Publisher · View at Google Scholar · View at Scopus
  36. T. Nishizaka, F. Nagatomo, H. Fujino et al., “Hyperbaric oxygen exposure reduces age-related decrease in oxidative capacity of the tibialis anterior muscle in mice,” Enzyme Research, vol. 2010, Article ID 824763, 2010. View at Publisher · View at Google Scholar
  37. F. Nagatomo, R. R. Roy, H. Takahashi, V. R. Edgerton, and A. Ishihara, “Effect of exposure to hyperbaric oxygen on diabetes-induced cataracts in mice,” Journal of Diabetes, vol. 3, no. 4, pp. 301–308, 2011. View at Publisher · View at Google Scholar