This study use BALB/c mice to establish the NEC model, which will be helpful for disease research on T cells. On day five, the two groups show no significant difference on body size. However, on day 10 the NEC mice were thinner than the control mice significantly.
The weight of the mice in the NEC group increased slowly, and the survival rate gradually decreased during the five days of model establishment. The NEC model could be advantageous for studying T cell polarization. Demonstrating the procedure will be Yan Tian, a postgraduate from my laboratory.
Gavage the mice with 20 to 30 microliters of LPS, and feed them with 40 to 50 microliters of formula on day five. From day five onwards, place them in a hypoxia device at 5%oxygen for 90 seconds, and re-oxygenate them for three minutes. Next, place the mice in a four degree Celsius environment for 15 minutes and transfer them to an incubator.
Closely observe all the mice, weigh them every day, record the survival of the mice during the induction period and record whether their stool is bloody and sticky. Holding the gastric tube in the right hand, fix the mouse's head with the index finger on the mouse's head gently pressed backward and downwards. Insert the gastric tube from the left corner of the mouth and slowly move it two to three centimeters to the center of the mouth.
Push 40 to 50 microliters of formula or 20 to 30 microliters of LPS into the digestive tract. If the mouse has a strong vomiting reflex and the gastric tube goes into the trachea, pull out gently and allow the mouse to rest before attempting gavage again. Immerse the fresh mouse illum tissue in 10%formalin, embed the tissues in paraffin and slice them of four micrometer sections.
To deparaffinize the sections, put them in xylene. Soak for five minutes successfully in absolute ethanol, 95%ethanol, 80%ethanol, 70%ethanol and distilled water. Next, stain the sections with hematoxylin solution for five minutes, and differentiate them in 1%hydrochloric acid and 70%alcohol for five seconds.
Finally, stain them with eosin solution for one minute, and examine the histopathology of the intestinal tissue at 40 times magnification. Photomicrographs of the intestinal pathology score from zero to four showed intact and normal mucosa, mild submucosal and lamina propria separation, moderate submucosal and lamina propria separation, severe submucosal and lamina propria separation, and intestinal villi disappearance with intestinal necrosis. The intestinal pathology score was significantly higher in the NEC group than in control with a P value of less than 0.001.
The stool was scored from zero with well-formed pellets to three with liquid stools. On day 10, the stool scores of the NEC groups were significantly higher in the NEC group with a P value of less than 0.001. On day five, the two groups showed no significant difference in body size.
However, on day 10, the mice in the NEC group were significantly thinner and smaller from head to tail than the mice in the control group. The weight of the mice in the NEC group increased slowly and the survival rate gradually decreased during the five days of model establishment. The resected ileocecal area of the intestinal tissue from NEC patients showed necrosis of intestinal mucosal tissue.
In this study, the mice in the NEC group also developed ileocecal hemorrhage and necrosis. When gavaging the mouse, ensure that the mouse does not struggle to avoid effecting the gastric tube insertion. Gavage the mice with 20 to 30 microliters of LPS and feed them with 40 to 50 microliters of formula on day five.
From day five onwards, place then in a hypoxia device at 5%oxygen for 90 seconds and reoxygenate them for three minutes. This model could be helpful for NEC disease research on T cells and might help study Th2 cell responses in NEC.
