BACKGROUND:A lot of work has been carried out on the development of the primary cultured rat myocardial cel s at home and abroad. The primary culture technology of rat myocardial cel s becomes more mature, but myocardial cel s from neonatal mice are not easy to be obtained under the same experimental conditions. The mouse genome has more similarities with the human genome, which has a higher research value. OBJECTIVE:To improve the primary culture method of neonatal mouse myocardial cel s, and to obtain myocardial cel s with high purity, vitality and original structure and function. METHODS:The mouse cardiac tissues were treated using an enzyme digestion method to isolate isolated single myocardial cel s:first, the cardiac tissues were digested using trypsin, and then col agenous fibers were treated with col agenase to isolate single myocardial cel s. The concentration and action time of trypsin and type II col agenase were adjusted, and the pH values of reagents and temperature of each step were strictly control ed. RESULTS AND CONCLUSION:At 24 hours after inoculation, the myocardial cel s began to be adherent;at 48 hours, independent pulsation of myocardial cel s could be observed;at 72 hours, myocardial cel s were cross-linked;and at 96 hours, myocardial cel s formed cel clusters and presented with consistent beating. The survival rate and purity of myocardial cel s were both over 95%. This modified method could successful y culture myocardial cel s with high purity and viablility from neonatal mice, and the structure and function of myocardial cel s could be retained. Therefore, it is a feasible culture method.

OBJECTIVEThe present study describes an improved in vitro culture method for obtaining high purity, vital and fully functional cardiomyocytes from neonatal rat.

METHODSAfter cutting ventricular tissue with improved method, ventricular tissues were digested with low concentrations of trypsin overnight at 4 °C, and then underwent collagenase II digestion. Thereafter, cardiomyocytes were purified by combined differential adhesion and chemical inhibition methods.

RESULTSAdherent cardiomyocytes were seen at 12 h after culture, spontaneously beating cardiomyocytes were observed at 24 h after culture, crosslinked cardiomyocytes were found at 48 h after culture, adhesion clustered cardiomyocytes were seen at 72 h after culture, dense network formed from inter-connected was evidenced together with radial arranged cell clusters and cell pseudopodia 96 h the mutual contact woven into and formed radically ordering cell clusters and island-like beating cardiomyocytes at 96 h after culture. The cell survival rate and purity were more than 98%.

CONCLUSIONFully functional spontaneous beating cardiomyocytes can be obtained by the use of this improved primary neonatal rat cardiomyocytes culture method.

Animals ; Animals, Newborn ; Cells, Cultured ; Myocytes, Cardiac ; cytology ; Primary Cell Culture ; methods ; Rats ; Rats, Sprague-Dawley

Objective: To evaluate the cardiac function of cecal ligation and puncture (CLP) induced sepsis rats with high-resolution ultrasound. Methods: According to the method of random number table, 48 adult male Sprague-Dawley (SD) rats were randomly divided into normal control group and sepsis 6, 12, 24, 30, 48 hours groups, with 8 rats in each group. The sepsis model was produced by CLP, and the rats in the normal control group were only anesthetized and resuscitated. The general situation after modeling in each group was observed, and the left ventricular function was assessed by high-resolution echocardiography at all the time points. The abdominal aorta blood of rats was collected, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and MB isoenzyme of creatine kinase (CK-MB) were determined by enzyme-linked immunosorbent assay (ELISA). The myocardial tissue was harvested, and the pathological changes in myocardial tissue were observed by hematoxylin-eosin (HE) staining. Results: The rats challenged to CLP displayed symptoms of sepsis, such as depression, ruffled fur, decreased diet and activity, and the symptoms became more obvious with the extension of time. High-resolution echocardiography could clearly show the structure of left ventricle in each group and obtain satisfactory M-mode echocardiography of left ventricle. The heart rate (HR) of rats in all sepsis groups was elevated with the increase in model time as measured by high-resolution ultrasound, and it was significantly higher than that in the normal control group at 12, 24, 30 hours (bpm: 359.66±23.33, 361.35±12.85, 392.67±11.33 vs. 306.24±29.79, all P < 0.05). Stroke volume (SV) and cardiac output (CO) in sepsis rats were decreased with the increase in model time, while left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were increased first and then decreased, and SV and LVEF in sepsis 48 hours group were significantly lower than those in the normal control group [SV (μL): 78.43±17.52 vs. 122.61±15.88, LVEF: 0.763±0.018 vs. 0.902±0.011, both P < 0.05]. Left ventricular weight (LVW) in all sepsis groups was increased to different degrees as compared with that in the normal control group, as well as the left ventricular anterior and posterior wall thickness increased in diastole and systole. Compared with the normal control group, the left ventricular posterior wall thickness was increased significantly at the end of diastolic and systolic period in the sepsis 12 hours group, and the left ventricular anterior wall thickness was also increased significantly at the end of diastolic period in the sepsis 48 hours group. The serum levels of TNF-α, IL-1β and CK-MB in sepsis rats were increased first and then decreased with the extension of model making time. The above parameters in the sepsis 48 hours group were still significantly higher than those in the normal control group [TNF-α (ng/L): 61.59±3.99 vs. 16.87±4.89, IL-1β (ng/L): 255.03±13.23 vs. 119.59±10.43, CK-MB (μg/L): 1.27±0.15 vs. 0.52±0.15, all P < 0.05]. HE staining showed that the myocardial striations of the rats in the normal control group were clear and complete, with normal morphology and orderly arrangement of cardiac cells. However in the sepsis groups, myocardial cells were swollen, ruptured and necrotic, and inflammatory cells were infiltrated, with myocardial fibers ruptured and necrosis dissolved, and the above pathological manifestations gradually increased with the extension of the model making time. Conclusion High-resolution ultrasound can evaluate the cardiac function of CLP induced sepsis rat model more comprehensive, and the consequence of evaluation index is consistent with the expression level of myocardial enzyme and histopathologic manifestations.