Endotracheal Intubation via Tracheotomy and Subsequent Thoracotomy in Rats for Non-Survival Applications

All animal activities described here have been approved by the institutional animal care and use committee (IACUC) of the Baden-Württemberg Regional Council in Karlsruhe, Germany. Experimental animals were managed as per institutional standards and according to German laws for animal use and care and according to the directives of the European Community Council (2010/63/EU) as well as the ARRIVE guidelines and were conducted in accredited facilities. Male Spraque Dawley rats from Janvier Labs with an order weight of 400 g were used.

1. Procedure preparation (Figure 1)

1. Prepare surgical preparation hooks by folding cannulas at an angle of 135° at 1 cm to the tip, connecting them to plastic perfusion tubes via Luer-lock, and applying tension using a surgical mosquito clamp (Figure 1A,B).
2. Prepare a rodent surgical exposure apparatus.
   NOTE: Besides the availability of commercial platforms, these can also easily be self-constructed cost-effectively, as in this study.
   1. For this, use a 40 cm x 50 cm steel plate with a 4 mm thickness. Create several 8 mm holes using a steel drill at the desired location for subsequent tension points. Place the Y-shaped fixation rods in these locations.
3. Place a heating pad on top of the steel plate of the surgical exposure apparatus in order to provide thermal support.
4. Stitch the surgical preparation hooks with attached plastic tubes and surgical mosquito clamps through the skin and use them to apply tension to the tissue for exposure (Figure 1C).
5. Remove the plug of a 10 mL plastic syringe with a male Luer-lock end and cut off the proximal end of the syringe. Insert the distal part into the ventilation bag as depicted and seal it airtight by covering it with a heat-shrinking tube and applying gentle heat using a laboratory torch (Figure 1D-F).
6. Shorten and bevel different sizes of intravenous catheters and insert a guide wire from an arterial leader-cath set with the flexible wire end at the tip into the catheter size most appropriate for the respective animal size (presumably 14 G) for subsequent Seldinger technique^22,23 during intubation (Figure 1G,H).

Figure 1: Experimental setup. (A) A folded cannula connected to a perfusion tube used as a surgical preparation hook. (B) Preparation hook with clamp positioned in a metal fixation rod. (C) Application of preparation hooks and metal stands for exposure of organs after laparotomy. (D) Ventilation bag from neonatology. (E–F) Airtight mounting of a Luer-lock syringe onto the ventilation bag using a heat-shrinking tube. (G) Shortened and beveled intravenous catheters in different sizes with a guide wire from an arterial leader-cath set for the Seldinger technique. (H) Final custom-built construction. Please click here to view a larger version of this figure.

2. Anesthesia and analgesia

1. Anesthetize the rat with the medication of choice.
   NOTE: Here, anesthesia and analgesia were established using ketamine 10% (100 mg/mL), xylazine 2% (20 mg/mL), carprofen 50 mg/mL, and isoflurane. A 1:10 dilution of xylazine and carprofen were used as stock solutions for application.
   1. Induce anesthesia by placing the animal in an induction box flooded with 100% oxygen at an inflow rate of 5 L/min complemented by 4% (v) of isoflurane using a vaporizer.
   2. After unconsciousness was achieved, remove the animal from the induction box and inject (i.p.) it with 100 mg/kg body weight ketamine and 4 mg/kg body weight xylazine.
      NOTE: This corresponds to 40 mg of ketamine (0.4 mL of the original solution) and 1.6 mg of xylazine (0.08 mL of the original solution or 0.8 mL of the 1:10 solution) for a 400 mg animal.
   3. Place the animal back into the isoflurane-flooded induction box until the effect of the i.p. injection sets in.
2. Guarantee proper anesthetic depth by testing the lack of response of the rat to a firm toe pinch.
3. Apply ophthalmic ointment to the eyes.
4. Assess and maintain the depth of anesthesia during surgery.
   1. When narcotic effects seemed to diminish over time (about every hour), inject (s.c) the animal with an additional 50 mg/kg body weight ketamine and 1 mg/kg body weight xylazine. This corresponds to 20 mg of ketamine (0.2 mL of the original solution) and 0.4 mg of xylazine (0.2 mL of the 1:10 solution) for a 400 mg animal.
   2. Provide additional analgesia before thoracotomy with an s.c. injection of 5 mg/kg body weight carprofen. This corresponds to 2 mg of carprofen (0.04 mL of the original solution or 0.4 mL of the 1:10 solution) for a 400 mg animal.

3. Surgical preparation (Figure 2)

1. Saturate the animal circulation with oxygen by offering inhalation of 100% oxygen via a transnasal neonatal mask (Figure 2A).
2. After 5 min, switch from the transnasal neonatal mask to the construction of a neonatal ventilation bag with a male plastic syringe Luer-lock tip as described above and offer oxygen in overflow with the syringe tip close to the animal nasal tip (Figure 2B).
3. Perform a median thoracocervical skin incision across the desired length and expose the surgical situs using the surgical preparation hooks (Figure 2C,D).
4. Advance the preparation by blunt dissection with fine scissors through the cervical fascia. Expose the sternocleidomastoid and infrahyoidal muscle and lateralize the median infrahyoidal muscle to the right after left lateral membrane dissection (Figure 2E-G).
5. Perform blunt dissection towards the trachea and tunneling of the trachea using overholt clamps (Figure 2H-I).
6. Sling the trachea using a silicone vessel loop and double sling the distal trachea using a polyfilament suture for later endotracheal cannula fixation (Figure 2J-N).

Figure 2: Preparation of the trachea. (A) Inhalation of oxygen via mask. (B) Switch from mask to male Luer-lock syringe for oxygen application. (C) Thoracocervical skin incision. (D) Preparation through the cervical fascia. (E) exposure of the sternocleidomastoid muscle and the infrahyoidal muscle. (F) Lateralization of the median infrahyoidal muscle. (G) Blunt dissection towards the trachea. (H) tunneling of the trachea using overholt clamps. (I) Slinging of the trachea using a silicone vessel loop. (J–N) Caudal double slinging of the trachea using a poly filament suture for later endotracheal cannula fixation. Please click here to view a larger version of this figure.

4. Intubation procedure (Figure 3)

1. Caudally lengthen the trachea using atraumatic pliers (Figure 3A,B).
2. Perform a partial incision of the trachea of 180° of circumference using scissors (Figure 3C-F).
3. Introduce a Seldinger guide wire with the flexible end into the trachea (Figure 3G-H) and subsequently introduce the modified intravenous catheter of appropriate girth into the trachea guided by the Seldinger wire (Figure 3I-N).
4. Remove the Seldinger wire (Figure 3O), connect the Luer-lock tip of the tracheal catheter to the modified ventilation bag, and start gentle lung-protective manual ventilation with high frequency and low tidal volume (Figure 3P). Perform manual ventilation as long as median thoracotomy has not been performed and the lung is not in plain sight.
5. Tie the previously placed locking suture with a sliding knot to avoid aerial leakage and accidental catheter removal (Figure 3Q-T).

Figure 3: Endotracheal intubation via tracheotomy. (A) Initial situation after preparation. (B) Caudal lengthening of the trachea using atraumatic pliers. (C–F) Partial incision of the trachea of 180° of circumference. (G,H) Introduction of a Seldinger guide wire into the trachea. (I–M) Introduction of the modified intravenous catheter into the trachea guided by the Seldinger wire. (N–O) Removal of the Seldinger wire. (P) Luer-lock connection of the tracheal catheter to the modified ventilation bag. (Q–T) Tying of the previously placed locking suture to avoid accidental catheter removal. Please click here to view a larger version of this figure.

5. Manual ventilation and median thoracotomy (Figure 4)

1. Start the median thoracotomy from the xiphoid using blunt and stable material scissors (Figure 4A,B) and continue cranially through the sternum. Always pause the ventilation for a few seconds when advancing with the scissors substernally and when cutting in order to avoid trauma to the lung (Figure 4C-E).
2. Gain thoracic exposure using surgical preparation hooks and remove mediastinal serosal membranes (Figure 4F-H).
3. Continue manual ventilation with desired frequency and tidal volume and expand monitoring (e.g., establishing an intra-arterial blood pressure measurement, installing a central venous or pulmonary arterial catheter depending on the purpose or respective research question) (Figure 4I-L).

Figure 4: Manual ventilation and median thoracotomy. (A) initial situation after intubation. (B–E) Median thoracotomy starting from the xiphoid.(F–I) Thoracic exposure using surgical preparation hooks. (J) Final setup during ventilation. (K–L) the process of manual ventilation. Please click here to view a larger version of this figure.

6. Euthanasia

1. Achieve euthanasia by abrupt sharp cardiectomy using scissors in the deeply anesthetized animal.