The overall goal of the following experiment is to be able to inject an agent of choice directly into the fetal mouse trachea, which allows for a more efficient delivery to the airways than existing techniques. This is achieved by exteriorizing the fetal head through a hyster otomy and accessing the fetal trachea. Next, a vertical neck incision is made to visualize the trachea and a needle is inserted under visual guidance.
Then fluorescent beads or a viral vector is injected into the trachea to efficiently deliver the substance of choice to the fetal airways. Microscopic analysis of tissue sections from treated lungs show that intra tracheal injection results in a more efficient delivery of fluorescent molecules or viral vectors to the fetal airways compared to the most commonly used intra amniotic delivery root. The main advantage of this technique compared to existing methods like fetal intra amniotic injection, is that you avoid the dilution of the injected substance into the amniotic fluid.
This maximizes the amount effect the particles present in the fetal mouse lung. In general, people new to this technology will find it extremely challenging to perform a perfect endotracheal injection. This is because the fetal trachea is extremely fragile and as such can be perforated very easily.
When you do this, you will perform a per tracheal injection and as such, fail to target the fetal lung. But as always, practice makes perfect In preparation for intra tracheal or it injection time amate pregnant and MRI mice. So they're 18 days pregnant at the time of surgery prior to and after surgery.
They're housed in filter top cages at normal room temperature and normal daylight with free access to water and chow. After anesthetizing the mouse with isof fluorine, place it on a heating pad at 37 degrees Celsius to maintain body temperature and check the level of anesthesia by performing a toe. Pinch, disinfect the abdomen with povidone iodine and perform a median laparotomy to expose the pregnant uterus, exteriorize the two uterine horns and count the number of gestational sacks.
One fetus per horn is operated on based on the optimal position for externalization of the fetal head. To fix the fetus inside the uterus from the shoulders passes six oh proline per string suture of approximately one centimeter in diameter through the uterine wall and the fetal membranes over the area where the fetal head will be exposed. Next, make an incision in the uterine wall inside the purse string of approximately 0.8 centimeters using small scissors with a sharp tip.
Next, gently squeeze the head and the neck of the fetus through the hyster otomy. Then gently pull the purse string, suture tight around the neck and use two micro mosquito forceps. To fix it in position use five o poly actin.
Nine 10 suture on two forceps placed into the mouth around the upper jaw to keep the fetal head in hyperextension under a stereo microscope. Visualize the trachea using sharp and blunt dissection to make a vertical neck incision further dissect the underlying tissue to reach the trachea. Using a 50 microliter Hamilton glass syringe with a 30 gauge sharp needle, inject a total volume of 30 microliters of substance.
Minimal influx of the injected liquid upon removal of the needle ensures that the injection was performed properly. To replace the head in the uterus, apply gentle pressure on the nose. The neck first goes back into the uterus followed by the head.
And finally, the nose. Insert a needle containing saline into the incision inside the purse string. Close the hyster otomy by tightening the purse string and inject a total of approximately 500 microliters of saline into the amniotic cavity to prevent oligo hydrus.
After replacing the uterus into the abdomen, use a five oh Vicryl running suture in two separate layers. To close the maternal abdominal wall, infiltrate the incision with 0.2%Xylocaine For postoperative pain relief. Keep the mouse on a heating pad until it has fully recovered to perform a fetal intra amniotic or IA injection.
After anesthetizing the pregnant mouse, use povidone iodine to disinfect the abdomen and perform a median laparotomy To expose the pregnant uterus, exteriorize the two uterine horns, and count the number of gestational sacks. Using a 50 microliter Hamilton glass syringe with a 30 gauge sharp needle, insert the needle into the sack, close to the fetus's mouth without coming in. Contact with the fetus and inject 30 microliters of solution into the amniotic fluid.
After all fetuses have been injected, reposition the uterus in the abdomen and use five oh Vicryl suture to the abdominal wall and skin. After euthanizing the dam, 36 hours after fetal intervention, deliver the fetuses by cesarean section and assess their viability by checking for the presence of a heartbeat, pink skin color and spontaneous movements. Place the pups nearby an oxygen supply to stimulate breathing and oxygen update.
Then mark the pups at the base of the tail. Using Chinese ink, a successful injection can be evaluated by euthanizing an operated fetus and dissecting the lungs to macroscopically, evaluate the presence of the visible dye in the lungs compared to non injected controlled lungs. Next, cover the remainder of the pups that meet the viability criteria.
Bedding material containing feces and urine from a foster mother with one day old pups and place the pups with the foster mother's litter. Leave no more than 10 pups in total in the foster mother's litter. Then place the cage in a quiet undisturbed environment for 12 hours and assess the survival rate of fostering to determine the optimal volume for it.
Injection 10, 20 and 30 microliter volumes of 100 nanometer red fluorescent molecules were empirically chosen. Shown here are confocal images of fixed lung sections with nuclei staining. Although at the time of fetal surgery a backflow was detected only after injection of 30 microliters indicating an excess of fluids injected 30 microliters gave the highest amount of fluorescent signal in the lung parenchyma as quantified by measuring the relative fluorescence as shown here, it injection resulted in a significantly higher delivery of fluoros spheres to the fetal lung compared to the I A root.
The gastrointestinal tract was also positive for both the IT and the IA injected animals. No red fluorescence was observed in other tissues from treated fetuses or in the negative control animals to assess the efficacy of viral transduction after injection, IT and IA injections were performed with RAAV vectors ENC coating firefly luciferase under the control of the chicken beta actin promoter at one week of age. The total photon flux for the IT group was significantly higher than for the IA pups and the negative control.
To distinguish between a correct and incorrect IT injection a combination of magnetic resonance imaging or MRI and bioluminescence imaging or BLI were carried out to obtain an overlay image which combines the surface. BLI signal with a visualization of the deeper anatomical structures from the MR imaging as shown here. At one week of age, BL imaging revealed a signal emanating from the neck and thoracic region.
Co-registration of MRI with BLI located luciferase gene expression in the pulmonary region following a correct injection but in the neck and abdominal area after an incorrect injection. Histological analysis by ex gal staining confirmed the in vivo co-registration. Once master the entire procedure of intra injection can be performed in half an hour from incision to closure of the mother animal.
A short operation time will result in better fetal survival and more animals that can be studied after birth.
