Gastrointestinal
Amelioration of Intestinal Ischemia–Reperfusion Injury with Intraluminal Hyperoxygenated Solution: Studies on Structural and Functional Changes of Enterocyte Mitochondria11

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Background

The aim of this study was to investigate the effects of intraluminal hyperoxygenated solution (HOS) on enterocyte mitochondrial structure and respiratory function after intestinal ischemia–reperfusion (IR) in rabbits.

Materials and methods

Thirty-two rabbits were divided randomly into four groups: control group in which sham operation was performed (Sham group), ischemia–reperfusion group (IR group), and two HOS treatment groups (H1 group and H2 group). Intestinal IR model was produced by clamping superior mesenteric artery (SMA) with an atraumatic vascular clamp for 1 h, followed by reperfusion for 2 h. Animals in the H1 group and H2 group received intraluminal HOS infusion for 1 h immediately after occlusion of SMA, and the rates of infusion were 10 and 20 mL/kg · h, respectively. After 2 h of reperfusion, enterocyte mitochondria morphological quantitative analysis was made with electron microscopy and biogenetics stereology, and the following parameters, including mitochondrial respiratory control ratio (RCR), intestinal O2 extraction ratio (ER), and mucosal ATP contents, were measured, respectively.

Results

After IR, the mitochondria was severely swollen with broken cristae, and mean transaction area, diameter, surface density, and volume density of the mitochondria increased significantly. Meanwhile, specific surface and numeral density of the mitochondria decreased significantly. The mitochondrial RCR, intestinal O2 ER, and mucosal ATP contents were all decreased significantly. There were no differences in all parameters between the IR group and H1 group. In the H2 group, the mitochondria were slightly swollen, and mean transaction area, diameter, surface density, and volume density of the mitochondria were all significantly lower, with the specific surface and numeral density of the mitochondria significantly higher compared with the IR group. The mitochondrial RCR, intestinal O2 ER and mucosal ATP contents in H2 group were all significantly higher than those in IR group.

Conclusions

Intraluminal HOS infusion at 20 mL/kg · h during ischemia ameliorates structural and functional changes of enterocyte mitochondria associated with intestinal IR injury, which is a safe, simple, and effective measure to protect the intestine from IR injury.

Introduction

Ischemia–reperfusion (IR) injury of the intestine is an important clinical event in disorders such as trauma, burn, septic or hypovolemic shock, strangulated hernias, neonatal necrotizing enterocolitis, mesenteric insufficiency, abdominal aortic aneurysm surgery, cardiopulmonary bypass, and intestinal transplantation [1]. Intestinal ischemia leads to depletion of cellular energy storages and the accumulation of toxic metabolites, resulting in cell necrosis [2]. Formation of toxic oxygen metabolites has been suggested to play an important role in the development of damage during intestinal IR injury. Studies have suggested that the xanthine dehydrogenase/xanthine oxidase (XO) system may be responsible for the generation of oxygen-free radicals during IR injury. Other studies using normal and XO-deficient animals suggested that during IR injury to the intestine, the invading phagocytes rather than XO form an important source of oxygen radicals [3, 4]. Reperfusion significantly exacerbates ischemia-induced mucosal injury via the formation of oxygen-free radicals, which cause biological damage by stimulating the free chain reaction known as lipid peroxidation, in which superoxide attacks the fatty acid side chains of the membrane phospholipids and causes organelle and cell disruption [2, 3]. This reperfusion damage frequently exceeds the original ischemic insult.

Among the internal organs, the intestine is probably the most sensitive to IR injury. The intestine is composed of labile cells that are easily injured by episodes of ischemia. Subsequent reperfusion of the intestine results in further damage to the mucosa [4, 5]. It has been shown that the enterocytes that are located at the tips of the villi are more sensitive to the insult of ischemia [6]. The initial site of abnormality in ischemia has been emphasized on the enterocyte mitochondria, which is particularly important in producing adenosine triphosphate (ATP) for cellular energy metabolism [7]. Anoxia is the most initial and important factor in the process of intestinal IR injury, and the mitochondria are more sensitive to anoxia than other organelles because mitochondria consume approximately 90% of the oxygen used by the cell [8, 9]. If oxygen is provided directly to the intestinal mucosa during ischemia period, the mitochondria respiratory function would be protected, and IR injury of intestine would be ameliorated.

In 1928, Hancock made a miraculous therapeutic effect in the treatment of septicemia with ultraviolet blood irradiation and oxygenation (UBIO) for the first time [10]. Subsequent researches have proved that UBIO also could improve oxygen-carrying capacity, regulate immunological function, and ameliorate microcirculation [11]. Chinese scientists derived inspiration from the achievements of UBIO and developed a kind of medical solution called hyperoxygenated solution (HOS) [12]. Using photochemistry techniques, oxygen can be dissolved largely in commonly used medical solutions (such as 5% glucose solution, normal saline, lactated Ringer’s solution, etc.); then, these solutions are turned into hyperoxygenated solutions, and the oxygen partial pressure (PO2) in these solutions can reach 750-900 mm Hg [12]. At constant temperature, the total volume of a gas that dissolves in a given volume of liquid depends on the partial pressure of the gas (P) and the solubility coefficient (Cs) of the gas in the liquid. This relationship is expressed mathematically in Henry’s law: Volume of dissolved gas = P × Cs × volume of liquid [13]. Because the PO2 in HOS is 9 to 10 times greater than that in arterial blood, it can be seen that HOS can “dissolve” approximately 9 to 10 times more oxygen than arterial blood at the same temperature. The administration of HOS in animal experiments has proved to preserve organs function and integrity after IR injury of the myocardium, spinal cord, and brain [12, 14]. The current study was undertaken to determine whether intraluminal infusion of HOS into the intestine of rabbits could preserve the enterocyte mitochondrial structure and respiratory function after intestinal IR injury and develop a new protective strategy against IR injury of the intestine.

Section snippets

Materials

Fig. 1 displays the schema of preparing HOS. Medical oxygen is introduced into the “medical hyper-oxygenated solution apparatus ” (Patent number: 922412936, Xi’an medical equipments Company, China) at an inflow of 3 L/min for 15 min. Treated with ultraviolet light (wavelength = 180–240 μm), some oxygen transforms into ozone. The oxygen/ozone mixture flows into the airtight base solution (5% glucose solution), and this base solution turns into HOS. The amount of oxygen dissolved in the water is

Histological Outcomes

Intraluminal HOS infusion at 20 mL/kg (H2 group) during the ischemia period protected intestinal mucosal integrity. However, intraluminal HOS infusion at 10 mL/kg (H1 group) failed to ameliorate intestinal mucosal damages associated with IR. Fig. 2 shows representative tissue sections from the four groups. The appearance of the H2 group sections in most areas was comparable with sham animals. Fig. 3 compares the mean ± SEM of the grading scores between the groups; IR (3.45 ± 0.71), H1 (3.18 ±

Discussion

The present study showed that intraluminal HOS infusion at 20 mL/kg during ischemia not only protected mucosal histology but also prevented structural and functional changes of enterocyte mitochondria associated with intestinal IR injury. Electron microscopic assessment revealed that enterocyte mitochondria from HOS-treated (20 mL/kg) IR animals virtually were indistinguishable from those of Sham group. Consistent with this observation, HOS treatment also ameliorated enterocyte mitochondrial

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    Supported by grant No.2002C2-18 from the Natural Science Foundation of Shaanxi Province.

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