A Mouse Fetal Skin Model of Scarless Wound Repair

The overall goal of the following experiment is to reproducibly model fetal cutaneous scarless wound healing in mice. This is achieved by first precisely timing the gestational age of embryos for fetal surgery using the superovulation technique. Next in utero, fetal surgery is performed to generate a fetal cutaneous wound at precise time points.

The results show that fetal fetal wounds generated at gestational age E 16.5 regenerate, whereas those at E 18.5 undergo repair with fibrotic scar based on histological staining. This method can help answer key questions in the scarless wound healing field, such as identifying molecular differences in early gestational wounds that heal without scar formation.Precisely. Timing the gestational age of mouse embryos for fetal surgery at E 16.5 and E 18.5 is of critical importance.

In this section, we detail our protocol for timing mouse pregnancies using PMS and HCG injections to induce super ovulation Between the hours of one and three o'clock in the afternoon. Begin by injecting female mice intraperitoneal with three to five units of PMS. In 100 microliters of PBS.

The females should be housed at five or fewer per cage. This is day one of the protocol. Next on day three between noon and 2:00 PM inject the same mice with HCG at the same concentration as the PMS injection.

Ovulation will occur at about 12 hours post injection immediately following the injections mate, these females with eight to 16 week old males, typically two females are mated to each male. Now on the morning of day four, between six and 10:00 AM separate. The females from the males record this time point as fetal age.

E 0.5 do not bother inspecting. For vaginal plugs, between 30 and 50%of the females will be pregnant depending on the strain for sterility. Firstly, clean all surfaces of the operating room and equipment with 70%isopropyl alcohol.

Secondly, sterilize the surgical supplies and instruments by autoclaving them. And thirdly, use sterile packs that include gauze and surgical instruments. After the pregnant dam at fetal age, E 16.5 to E 18.5 has been anesthetized.

Confirm a surgical plane of anesthesia using a toe pinch. Then apply ophthalmic ointment to the eyes and depilatory cream to the abdomen. Remove the cream within 30 seconds and then scrub the skin with povidone iodine and alcohol.

Now under a dissection microscope, perform a midline laparotomy using microsurgical scissors. Gently expose the uterus and the fetuses and irrigate the surgical field with warm PBS from a blunt tip needle. A blunt tip needle can be made by bending a normal needle.

Now position a fetus so there is full access to the dorsum. Then using 7.0 nylon suture. Pass a purse string stitch through the uterus, overlying the site of intended dorsal wounding.

Position the purse string over a region of dorsum to the left or right of the spinal cord, and in a region of uterine wall devoid of large blood vessels. Next in the middle of the purse string, make a three millimeter incision through the uterine wall and the amniotic sack. Follow this incision by irrigating with more warm PBS.

Then in the dorsum of the fetus, use microsurgical scissors to cut a single full thickness excisional wound about one millimeter long. Clean up this incision with a cotton tip applicator. Now inject three microliters of India ink subcutaneously into the wound site to market's location.

Follow the injection with warm PBS. Make sure the ink is retained in the wound site. The next few steps require an assistant while closing the purse string suture.

Have an assistant sustain a stream of warm PBS ejected from a blunted 10 gauge syringe into the amniotic sack. As the suture strings close, retract the syringe. Now gently return the uterus into the abdominal cavity, avert the skin and peritoneum, and have the assistant irrigate the abdominal cavity.

Finish by placing staples in the skin and peritoneum to quickly close the abdominal cavity. After closing the abdomen, place the animal in a warm incubator Set at 37 degrees Celsius. Observe the recovery over the next 30 minutes or until the animal has regained stable sternal recumbent.

Then administer subcutaneous injection of buprenorphine and house the animal individually with free access to food and water. Do not house the animal with others until it has fully recovered from the procedure. Keep providing buprenorphine every 12 hours over the next 48 hours post-surgery and as needed based on regular pain assessments.

If necessary, additional postoperative pain relief can be provided with carprofen 48 hours post-surgery. Sacrifice the mother with an overdose of isof fluorine. Sustain the exposure to isof fluorine for one minute after she stops breathing.

Confirm the euthanasia with a cervical dislocation and harvest the wounded fetus and an UN wounded control fetus for histologic analysis. Fix the embryos in 4%PFA, followed by embedding in paraffin. For fluorescent transgenic models.

Cryo-preservation with OCT compound may be appropriate. After performing the procedure correctly, het toin and eosin staining reveals that E 16.5 skin heals with minimal scarring, complete reepithelialization, and only a small increase in the number of inflammatory cells. Moreover, these wounds heal with a normal skin architecture and contain regenerating hair follicles within the site of injury.

As noted by the arrows, an ink depression at the arrowhead is normal when the ink is applied correctly. Finally, tri chrom staining reveals a fine reticular dermal collagen pattern characteristic of unscarred dermis compared to wounds made at E 16.5. Wounds made at E 18.5 have a dense scar with loss of normal skin architecture at the site of injury.

This is readily revealed by h and d staining. Similarly, tri chrome staining of E 18.5 wounded embryos reveals a dense pattern of disorganized collagen deposition as noted by the arrows While attempting this procedure, it is important to remember to be gentle when manipulating the uterus and fetus.