| Total hip replacement and femoral nailing are widely accepted operations. The complication of pulmonary fat embolism syndrome would lead to death if it occurred. Recent research showed that pulmonary fat embolism existed in almost all such patients. But why only some patients developed clinical symptoms remains unknown. For further studying we established an animal model on rats and further probe for its pathological changes.Materials and methods1. Animal model establishment80 male Sprague-Dawley SPF rats were selected, weighing 350~410g (12-14 w) (purchased from The Animal Facilities of Akita University, Japan). Rats were randomly divided into shock-reaming, reaming, shock and control groups. Strictly selected satisfied rats, shock-reaming (7), reaming (8), shock (7) and control groups (5). Rats were fasted 12h before experiment and water was taken freely. The whole procedure was under sterile conditions. Pentobarbital were injected abdomently and 15 min later second injection was given through penile vein. Central incision was taken and canulation was taken to left carotid artery and right jugular vein. Bleeding were restricted less than 0.5 ml. Left canula was connected with blood pressure monitor and right canula was connected with transfusion device to supply for the fluid loss and keep maintaince of venous anesthesia. Total fluid was about 14-16ml. Heparin was given in flushing fluid. The incision was sutured with double layers and canula was fixed. The experiment started when blood pressure was stable 30 min later.At 0 min, shock-reaming and shock groups started bleeding through carotid artery for 10 min, making the blood pressure reaching SOmmHg. The blood was kept in refrigerator at 4癈.Then rats were in natural recovery for 50min. At 60 minthe original blood which was taken out and warmed 10 min earlier was transrused back (lasting 10 min). The recovery lasted until 120 min while shock-reaming and reaming groups started reaming and intramedullary fixation.Rats were in lateral position. Small incision of 1cm long was taken on bilateral intertrochanteric fossa. Tendons were cut with sharp knife and reaming was finished with 1.6mm drill and 1.2mm nail was driven in. Double layer suture was taken. Standardized experimental time was 255 min. Blood gas analysis were taken at the time of 0, 60,120,150,165,195 and 255 min. Blood routine test was taken at 0 and 255 min. At 255 min, animals were killed with Pentobartbital. Left lung was resected and preserved in liquid nitrogen for MPO (Myeloperoxidase) measurement. Right lung was injected with 20% formalin through trachea, resected and preserved in 20% fromalin. X-ray was taken to exclude fracture occurrence. Control animals had the same procedure except for the shock and reaming processes. The lung samples were given HE staining and examined microscopically. And lung injury score was valued semiquantitively under 100 time's microscope for whole fields of each slide of all samples. 2. MPO measurementLungs were externally rinsed with saline, blotted dry, and separately weighed in preparation for MPO assay. Lung tissue was homogenized for 30 s (Virtishear homogenizer;) in a 4-ml 20 mM potassium phosphate buffer, pH 7.4, and centrifuged for 30 min at 40,OOOg,4癈.The pellet was resuspended in 4ml 50 mM potassium phosphate buffer, pH6.0, containing 0.5g/dl cetrimonium bromide. Resuspended pellet were frozen at -80 癈 until the MPO assay was performed. Frozen samples were thawed, sonicated for 90 s at full power (ultrasonic disrupter), incubated in a 60 癈 water bath for 2 h, and centrifuged for 10 min at maximum speed (Becman Microfuge) Supernatant, 0.1 ml, was added to 2.9 ml of 50 mM potassium phosphate buffer, pH 6.0, containing 0.167mg/ml 0-dianisidine and H2O2; absorbance of 460 nm visible light was measured for 3 min (Beckman DU7 spectrophotometer) MPO activity per gram wet lung (gwl) was calculated byMyeloperoxidase activity (tJ/gwlHA460)(13.5)/lung weight(g)Where A460 is the change in absorbance of 460 nm light from 1- to 3-min... |
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