Hi, I am Aji Kumar Gs from Cardiovascular Disease Biology Division Raj Gandhi, center for Biotechnology India. I am working under the guidance of process karta and in this video we will demonstrate a step-by-step procedure for ascending I OTE construction in rats. In our laboratory, we utilize this annual model to study the pathophysiology and underlying molecular mechanisms in cardiac hypertrophy and progressive heart failure.
We also use this model to study left to heart failure associated pulmonary hypertension. So let's get started And aestheticize the animal and keep it in supine position. Perform endotracheal intubation by making a halfway incision between two tracheal rings and inserting the tracheal cannula, which is in turn connected to a rodent ventilator.
Then make a skin incision of about two centimeter long at the left chest region between second and third ribs and separate the muscle layers. To visualize the iota constriction of the ascending portion of the iota is to be carried out using a small sized titanium clip with an applicator, oppose the ribs and then suture the muscle layers and skin in an interrupted pattern. After the removal of endotracheal tube, close the trachea muscle, layers and skin and allow the animal to recover.
The procedure was done after getting approval from institutional animal ethics committee. Prior to the surgery, disinfect the operating field with 70%alcohol and keep the heating pad aseptically. Keep the rodent ventilator and anesthesia unit ready for surgery.
Sterilize the surgical instruments by autoclaving. Major surgical tools for ascending I otic constriction are sterile gloves, thumb forceps, scissors, surgical blades, small size titanium clips and applicator artery forceps with gauze in case of bleeding, a needle holder proline suture material, braided silk suture and needle. Maintain the rat under an antibiotic umbrella for 24 hours and aestheticize the rats in an induction chamber with 3%ISO fluorine mixed with a hundred percent oxygen.
Remove the fur from neckline and left chest region of the rat and keep the animal at top. A heating pad. Maintain the anesthesia with 1.5%ISO fluoride check pedal reflex for the confirmation of successful anesthesia.
Disinfect the surgical area with povidone iodine solution followed by 70%alcohol. Repeat the procedure three times. Place a sterile drape over the animal exposing the surgical site only.
Make a skin incision parallel to trachea and separate the underlying tissue. In order to expose the trachea. Apply a tie of silk thread underneath the trachea and gently raise it upwards.
Make a halfway incision between two cartilage rings and insert the tracheal canula. Connect the tracheal canula to a rodent ventilator. The ventilator is to be set at a respiratory rate of 50 breaths per minute.
Tidal volume 1.7 ml and inspiration times 0.6 seconds. Make an Incision at the left chest region between second and third ribs. The intercostal muscles are to be separated layer by Layer.
Care should Be taken to avoid any injury to the lungs while making the incision between the ribs. After retracting the ribs, carefully locate the ascending portion of the iota by gently manipulating the fat tissue. The arrow indicates ascending portion of the iota.
Place a small size titanium clip around the ascending iota with the help of an applicator, thereby constricting the iota 50 to 60%of the Original diameter. Following I otic Constriction, reposition the heart and approximate the ribs with proline suture Simultaneously Pause the ventilator for about two to four seconds in order to reinflate The lungs. Post the Muscles in layers with proline, followed by skin with silk sutures in an interrupted Suture pattern.
Gradually Reduce the respiratory rate set at the ventilator and observe the animal for spontaneous breathing. Remove the Tracheal cannula once spontaneous and regular breathing was reestablished. Now anesthesia is maintained through nose cone.
Close the opening between tracheal rings with proline sutures. Close the muscle layers and skin with proline and silk sutures respectively. Apply povidone iodine solution over the Surgical area as postoperative analgesia inject.
Tremadol intraperitoneal saline solution can be administered intraperitoneal. If signs of dehydration appear gradually lower The anesthesia to off position after the animal Recovers, shifted to the cage and administer amoxicillin orally mixed with drinking water for Seven days. The intensity Of pressure overload produced by constriction of ascending iota can be determined by observing the pressure gradient across the I otic constriction site by two dimensional echocardiography.
Left panel of the figure shows Doppler echo of aortic constricted rat having a pressure gradient of 63 mm of mercury. And the right panel represents a sham controlled rat without significant pressure gradient across the iota. The top panel shows M mode echocardiography of left ventricle in rats, eight weeks post-surgery, and bottom panel represents sham control, interventricular septal thickness and the left ventricular posterior wall thickness were significantly increased while left ventricular internal diameter was significantly reduced in rats undergone I otic constriction.
Compared with sham operated control groups, a significant cardiac hypertrophy is observed in rats undergone ascending I otic constriction post eight weeks of surgery compared to sham operated control groups. Hi, I am Raj. Here we explain the procedure for constriction of ascending iota in rat.
The main advantage of this model is gradual development of cardiac hypertrophy and heart failure. Similar to clinical situation in chronic hypertension in this mother care should be taken in the application of Titanic clip for constriction of the iota. Most important thing is that the application of Titanic clip should be sufficient to produce critical iota constriction for left 20 plus stress, but not severe enough to produce acute electronic failure and pulmonary edema and care should be also taken to avoid any injury to the lungs while making incision between the ribs.
The ventilators should be posted for three to four seconds while opposing the ribs in order to reinflate the lungs. So thank you for watching this video and good luck with your experiments.
