The most important advantages of this technique are reversibility, reduction in cost of the animals and maintenance, and a shorter pregnancy period. Moreover, animal mortality is reduced because there is no need for hysterectomy. And there are wider opportunities for genetic workup.
This technique can be used for investigating the pathophysiology of the congenital diaphragmatic hernia. It can also be applied to understanding other diseases in the lung, such as cows and pulmonary hypoplasia. Begin by allowing the mice to mate in the same cage from 6:00 PM to 9:00 AM the next day.
To determine E0, observe the vaginal plug which has a homogenous, outer zone attached to the vaginal wall and an inner zone that is fibrous and include some spermatozoa that form entangled masses mixed with the fibers of the plug material. Record the weight of the mice at the time of mating and on E10 to ensure ongoing pregnancy and perform the surgery at E16.5. Sterilize the instruments that are going to be used during the surgery.
Preheat the surgery platform to 24 degrees Celsius and prepare warm saline prior to surgery. Create a warm environment for recovery and leave wet food inside the cage for the early feeding. Clean the abdominal surface with alcohol and povidone iodine and maintain sterile conditions throughout the operation.
Perform a vertical incision for the laparotomy of pregnant dams and cut all layers separately. Identify the uterine horns on each side and determine the candidate fetuses for the surgery. Operate on two fetuses in each uterine horn if there is an even number of fetuses on each side.
And on one fetus in each uterine horn if there is an odd number. Using 2X magnification glasses for visualization, position the uterine horn in a transverse fashion. Position the pups facing upward between two fingers using the eyes and the tail as a guide.
Apply gentle pressure to the pup's head to allow extension of the head and visualization of the neck. Perform tracheal occlusion or TO using an atraumatic needle and a 6 by 0 polypropylene suture. Keep the placenta on the side and far from the entrance and exit points of the needle.
Insert the needle transversely through the side of the uterus, away from the placenta through the one-third anterior part of the neck. Then move the needle gently to the midline of the neck. Direct it to the anterior part.
Then exit the neck between the trachea and opposite the carotid sheath and uterus. Knot the suture, taking care to maintain the integrity of the membranes and uterine wall. Replace the uterine horn in the abdomen and inject two milliliters of warm sterile saline into the peritoneal cavity before closure.
Place a running 5 by 0 polyglactin suture to close the abdominal wall. And close the skin with a non-running silk suture. Apply 0.1 microgram per kilogram of buprenorphine intraperitoneally for analgesia and allow the dam to recover in a warm incubator.
Observe that the operated animal can feed itself and keep it alone in its individual cage. Harvest all the fetuses by E18.5 by ceasarean section and check their viability by watching their movements. Weigh all the fetuses.
Perform a vertical incision on the thorax for a thoracotomy to dissect the lungs. And weigh them to calculate total lung to body weight ratio. Snap freeze the tissue in liquid nitrogen, optimal cutting temperature compound, and dry ice.
Cut the samples in sections of 10 micrometers using a cryostat, and mount them on polylysine-coated slides. Bake the slides overnight at 60 degrees Celsius and stain the baked slides with hematoxylin and eosin. Image of the slides at 10 to 20X magnification, using a widefield microscope.
For protein and DNA analyses, snap freeze the tissue and homogenized it in 300 microliters of radioimmunoprecipitation assay buffer. Centrifuge at 18, 000 x g and 4 degrees Celsius for five minutes. Then extract and quantify protein, DNA, and RNA.
The mean body weight, lung weight, and LBWR were higher in the TO group than in the control group. Lung DNA amounts and the DNA to protein ratio were higher in the TO group. No difference was observed in lung RNA, and protein amounts were lower in the TO group than in the control group.
Histological analyses of the E18.5 control lungs showed the late or early saccular stage of lung development with developing airspaces and thickened interstitium between epithelial surfaces. The lungs in the TO group had dilated central and distal airspaces with subjectively higher numbers of nuclei. When attempting this protocol, We fix the sutures in a way that the eyes of the fetus are visible and the neck is extended so that the needle does not disrupt the adjusted structures.
The removal of the structures to the rebirth of the fetus in the nitrile fan and knockout models of CDH will be the future applications of this technique.
