| Objective: Many severe limb trauma patients if not to obtain promptly effective resuscitation therapy, only gained delayed resuscitation, seldom got anticipatively therapeutic effect and only obtained limb ischemia reperfusion (IR) injury as well as high incidence of shock. Furthermore, the patients would get high incidence of multiple organ dysfunction syndrome (MODS) and mortality in post-course of disease. Kidney is an organ with high perfusion and rather sensitive to ischemia and reperfusion. There are many factors for IR injury. Apoptosis induced by inflammation cascade reaction and oxidative damage are the two main factors. Carbon monoxide (CO) has important functions under physiological and pathological conditions. Recent studies suggested that CO has anti-inflammatory and anti-apoptotic effects. According to this, many researchers propose a bold suggestion that using light concentration CO to prevention and cure IR injury. Our laboratories have demonstrated that the administration of exogenous carbon monoxide (CO) at the volume fraction of 0.05% could prevent MODS in a model of IR injury on both side hind limbs. However, the indications and contraindications have not yet been investigated, especially to the severe limb trauma patients who would have generated shock caused by excessive compression and blood loss.Methods: Fifty-six healthy male Sprague-Dawley rats were selected and randomly divided into three groups: IR (twenty-four rats), RC (twenty-four rats), S (eight rats). SD rats underwent anesthesia and bilateral femoral artery occlusion. The roots of bilateral hind limbs were banded by belt. Rats were maintained at this state for 8 hours, and then showed symptom of shock. With the reperfusion for 6 or 8 hours, the kidney was taken out for detection. Rats in sham group underwent sham IR injury. Additionally, sham animals were not ?ischemia. 6 hours after the operation, RC group was exposed to medical air mixed CO at the volume fraction of 0.075% continuously until 4 hours after reperfusion. It's 6 hours totally. S and IR groups were exposed to normal air for 6 hours continuously. Each 8 rats in IR and RC group were used to observe blood pressure and 10 days survival rate. 8 rats in IR group, 8 rats in RC and 4 rats in S group, were killed after 6 hours of reperfusion. 8 rats in IR group, 8 rats in RC group and the other 4 rats in S group were killed after12 hours of reperfusion. From right atrium, 5 ml blood was harvested. Serum potassium ion (k+), creatinine (CRE) and blood urea nitrogen (BUN) value were tested to detect renal function. The left ischemia kidneys were harvested and used for wet/dry weight ratio measurement and general observation. Renal color, resilience, tumescence, effusion and adhesions were observed and taken pictures. The electronic analytical balance was used to measure the wet weight of severed kidneys. After then, at 60℃, the kidneys were dried for 7 days in electrical thermostat. After the kidneys were heated to dryness, dry weights of the severed kidneys were measured. The wet/dry weight ratio was thus calculated. Kidney tissue morphological injuries were analyzed by the HE staining.Results: (1) The 10 days survival rate: The blood pressure of caudal artery in IR group started to lose immediately after reperfusion. The process was divided into four phases. The first one was from the reperfusion to 2 hours later, during which the average pressure fell rapidly and dropped to 8.6708±0.5992 kpa in the end of this phase. In the second phase, from 2 to 4 hours after reperfusion, the range of the falling pressure was small. The third phase was from 4 to 8 hours after reperfusion, during which the average blood pressure fell slowly and dropped to 5.3259±0.3832 kpa in the end. After 8 hours of reperfusion until the death belonged to the fourth phase, during which the pressure kept at a low level about 5kpa. Animals were dying state. 6 rats in IR group died during 13 to 14 hours after reperfusion. The other 2 rats died 20 hours after reperfusion. 10 days survival rate of IR group was 0. Contrastively, in RC group: the blood pressure of caudal artery fell slower with smaller range. ?Its pressure dropped quickly in the first phase, but declined by a littler margin compared to IR group. After reperfusion for 2 hours the average pressure fluctuated to 10.3843±1.3111 kpa. There were significant reductions on both velocity and range of the falling pressure in the third phase. The pressure dropped to 8.3300±0.4224 kpa after 8 hours of reperfusion. It's worth noting that the average pressure did not keep falling and started to rise placidly in the fourth phase. The average pressure reached to 9.4157±0.3305 kpa after 13 hours of reperfusion. One rat died after reperfusion for 20 hours. The other seven rats were still alive and doing fine after reperfusion for 10 days. 10 days survival rate was 7/8. (2) Observed by plasma k+, CRE, BUN: The result of S group was the lowest with normal plasma k+, CRE and BUN. However, the plasma k+, CRE, BUN in IR group became the highest. RC group was in the middle. There was significant difference between S group and IR group (P<0.05). There was significant difference between S group and RC group (P<0.05). Moreover, the plasma k+, CRE, BUN in RC group decreased significantly when compared with the IR group (P<0.05). (3)The wet/dry weight ratio: IR group was the highest. S group was the lowest and RC group was in the middle. There was no significant difference between S group and RC group (P<0.05). The wet/dry weight ratio in RC group decreased significantly when compared with IR group (P<0.05). (4)The observation on the whole: The kidney of S group showed the color of dark red and normal form. There were no hemorrhagic effusion and necrotic tissue. While the kidney of IR group showed the color of brown and edema, while dysfunction of elasticity appeared obviously with plenty of hemorrhagic effusion under the capsule. These changes based on IR 12 h ones were more serious than IR 6 h ones. Compared with group IR, the pathological changes were slight in RC group. However, the color and edema of kidney in RC Group became improved. Hemorrhagic effusion of RC group appeared much less and the color turned dark red. These changes were more obvious in RC 12 h ones than RC 6 h ones. (5) Observed by HE staining: The result of the S group showed that the kidney tubules arranged orderly. The shape of kidney capsule and ?nuclei remained normal. Coloration was symmetrical. The nucleolus was clear. And there were no inflammatory cells infiltration. In IR group, the degeneration and necrosis were significant in kidney tissue. The kidney tissue revealed the infiltration of inflammatory cells and wide hemorrhage. The incarcerated cells were increased and capillary lumen became narrow with fatty casts. Kidney tubules arranged derangement and there was more vacuolization in IR group. Renal tubule epithelial cells were swelled obviously. Cytoplasm swelling and apoptosis could be found. The cytoplasm dissolved. These pathological changes based on IR 6 h ones were more serious than the IR 12 h ones. In RC group, there were little pieces of cells showing degeneration and dissolved. Most cells showed slight swelling and focal necrosis. Coloration of the cytoplasm and nucleus were close to normal. CO treatment reduced the number of infiltrating macrophages. Only a few inflammatory cells were found in kidney tissue. Apoptosis was not observed and hemorrhages decreased after the treatment. The pathological changes in RC 12 h ones were more lightened than the RC 6 h ones.Conclusions: These results demonstrate that exogenous low-dose CO treatment can attenuate kidney injury induced by serious limb IR injury in rats.
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