Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

The overall goal of this procedure is to surgically induce intrauterine growth restriction or IUGR in a rabbit fetus and examine the fetal cardiovascular response to placental insufficiency. This is accomplished by first performing a midline laparotomy at gestational age day 25 and carefully exposing the rabbit by cornew at uterus next 40 to 50%of the uteroplacental vessels supplying each gestational sack in one uterine horn are surgically ligated, and the fetuses in the second uterine horn are left as controls. At gestational age day 30, a repeat midline laparotomy is then performed using an intravenous infusion of ketamine and xylazine anesthesia to minimize fetal cardiac effects.

Finally, fetal placental doppler ultrasound and fetal echocardiography are carried out to obtain measurements of fetal hemodynamics and cardiac function and structure in IUGR and control fetuses. Ultimately, results can be obtained that show the fetal cardiovascular response to increased placental resistance through biometric analysis, fetal placental doppler, ultrasound, and novel fetal echocardiography. This method can help answer key questions in fetal medicine and adult cardiology, such as whether abnormal physiological pressures can lead to disproportionately profound changes in cardiac structure and function later in life.

The implications of this technique extend towards therapy for IAGR because after surgical induction of IGR, this model allows sufficient time for the researcher to then administer potential therapies such as anticoagulants, antioxidants, and glucocorticoids, and then to study their effect on fetal cardiac function, but later in gestation, The experiment will be demonstrated by Dr.Ryan Hodges, obstetrician and postdoctoral researcher. Together with Andrea La Gersh, a cardiologist and postdoctoral researcher, together with doctors Andrea Mge and Philip Deko obstetricians in training and doctoral researchers at this department. Prior to this surgery, use randomization software to randomly allocate a case IUGR and a control uterine horn in the bicornuate rabbit uterus for time, dated pregnant rabbits from gestational age or GA 18 to 25 days, house them in individual cages on a 12 hour light dark schedule with access to water and standard rabbit chow ad libido at ga 25 days, anesthetize the rabbits by administering a hind leg intramuscular injection of ketamine and xylazine.

Maintain anesthesia through inhalation of one to 4%isof fluorine in oxygen at two liters per minute. After shaving the maternal second and third toe, place a pulse oximeter to continuously monitor heart rate and arterial oxygen saturation prior to beginning surgery in a single 2.5 milliliter syringe subcutaneously, administer 300, 000 IU of penicillin, G 0.9 milligrams per kilogram medroxyprogesterone acetate, and 0.3 milligrams per kilogram.Buprenorphine. Place the rabbit on a warming pad, remove the hair from the surgical field and apply povidone iodine to disinfect the area.

Next subcutaneously, administer local anesthetic around the incision site, which is about five centimeters in length at the midline along the lower two thirds of the abdomen. Then perform an abdomen pinch to verify that the animal is sedated with the scalpel. Open the skin.

Then use a combination of sharp and blunt dissection to expose the rectus sheath. Taking care to avoid the maternal mammary glands located on either side of the midline. Grasp the rectus sheath and peritoneum tent the tissue to avoid the abdominal contents and use sharp dissection to carefully enter the abdomen.

Surround the surgical field with gauze drapes soaked in warm saline before carefully grasping and exteriorizing both sides of the bicornuate uterus to prevent pain, bleeding, and uterine contractility. Take care not to introduce tension on the vaginal or ovarian ends of the uterine horn. Use warm saline to continuously irrigate the exposed uterus.

Palpate and record the number of fetuses in each horn, and after identifying the previously allocated case horn for IUGR induction, return the control horn into the abdomen. Next in the case horn, identify the uteroplacental vessels to each gestational sack. Then using a five aught poly lackin suture ligate, 40 to 50%of them or the largest vascular bundle if there are multiples such as three.

When the ligations are complete, irrigate the uterus with warm saline and carefully return it to the abdomen. Use two OTT poly clain suture to close the abdomen in two layers, and use three OTT poly clain subcuticular sutures to close the skin. Finally, discontinue anesthesia and closely observe the animal for recovery of respiration.

House the rabbit in a private cage with access to food and water ad libido, and observe daily at GA 30 days. Place the rabbit on a warming pad for echocardiographic and ultrasound evaluation using the visual sonics vivo 2100 high resolution micro ultrasound platform with cardiac and strain analysis software. The system uses the visual sonics MS two 50 transducer for data acquisition.

It has a center frequency of 21 megahertz with a bandwidth of 13 to 24 megahertz. The geometric focus is 15 millimeters. The maximum image width is 23 millimeters, and the maximum image depth is 30 millimeters.

The footprint is 28 by 5.75 millimeters. Data is acquired in accordance with the American Society of Echocardiography guidelines and standards for performance of the fetal echocardiogram. After anesthetizing the rabbit as demonstrated earlier in the video, insert a 24 gauge cannula into a peripheral auricular vein to obtain intravenous access.

Maintain anesthesia with a continuous infusion of ketamine and xylazine in normal saline via a syringe pump at 40 milliliters per hour. Secure a three-way tap and attach a five milliliter syringe of rescue anesthesia and deliver a subcutaneous injection of buprenorphine. Apply a mask and deliver two liters per minute of oxygen and place a pulse oximeter on the second and third toe.

Once the abdomen has been opened as demonstrated earlier, and the rabbit is placed in a lateral position facing towards the researcher, expose a single gestational sack and place it on gauze irrigated with warm saline to avoid environmental and iatrogenic effects on fetal cardiac function, immediately begin ultrasound examination working in a systematic and efficient manner at the level of the lateral four chamber view Record a B mode cine loop of at least five cardiac cycles to determine strain, strain, rate, velocity, and displacement. Then still working in this standard view, obtain M mode echocardiography indices. Next at the level of the apical four chamber view, repeat the B mode cine loop and use M mode echocardiography to assess tricuspid and mitral annular plane systolic excursion and longitudinal strain and strain rate.

This apical view also permits assessment of valvular flow velocity wave forms, and the modified myocardial performance index. To obtain flow velocity, wave forms or FVW of the umbilical artery, use color doppler to locate the umbilical vessels and place the pulse doppler sample gate over the umbilical artery on a free loop of umbilical cord. Once the circle of Willis is located, find the cerebral artery FVW by placing the pulse Doppler sample gate just beyond the origin of the middle cerebral artery or MCA.

To obtain the ductus OSIS or DV FVW using either a sagittal or transverse view of the fetus, place the pulse doppler sample gate at the proximal portion of the DV where it originates from the intrahepatic umbilical vein. To analyze the data offline, use the visual sonics cardiac package for calculating M mode echocardiography indices, and for strain analysis, use the vivo strain algorithm as previously described. Finally, using the visual sonics cardiovascular analysis software, calculate the puls utility index or PI show.Shown.

Here is an asymmetrical growth restricted fetus and placenta from uteroplacental vascular ligation as compared to a normal control fetus and placenta. Asymmetrical growth is confirmed by reduced neonatal birth weight and an increased head circumference to abdominal circumference ratio compared to controls as seen here with results from fetal placental doppler. A control fetus exhibits a normal low resistance pattern of positive and diastolic flow with progressive increases in placental resistance found in IUGR fetuses absence, and then reversed end diastolic flow is apparent.

This figure demonstrates a normal high resistance middle cerebral artery doppler signal in a healthy fetus and a positive a wave ductus osis. In the same fetus in IUGR fetuses, an increased pulsatility index of the ductus osis and reversal of the A wave can be seen. Representative M mode fetal echocardiography results are shown here.

This lateral view allows calculation of internal ventricular diameters and volumes. The apical view allows calculation of tricuspid and mitral annular plane systolic excursion. This figure shows speckle tracking of velocity vectors and the resultant regional strain curves to calculate strain, strain rate, displacement and velocity.

While attempting this procedure is important to remember the invasive nature of experimental fetal surgery, the uterus and the fetus must be handled with care. All of the measures we have described to reduce uterine activity and prevent fetal bradycardia during echocardiography are all highly recommended to ensure that you'll have successful experiments Following this procedure. A methods like various tissue analysis from the placental and fetal tissues can be performed in order to answer additional questions like, what are the fundamental mechanisms involved in fetal cardiac adaptations in IGR?

After having watched this video, you should have an idea how to induce fetal growth restriction in the rapid model and how to assess by ultrasound using doppler and fetal echocardiography. Its effects.