All the procedures were approved and performed in compliance with the regional ethics committee (protocol (#17858 and #32499, CEEA-Pays de la Loire, France) according to Directive 2010/63/EU of the European Union. The reporting is in accordance with current ARRIVE guidelines and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 2011).

1. Ethical status and general information on rats

1. Male Wistar Han rats (Charles River, Saint-Germain-Nuelles, France) were delivered to the Unité Thérapeutique Expérimentale at 10 weeks of age. House the rats for at least 1 week under standard temperature (21-24 °C), humidity (40%-60%), and 12 h light/dark cycle with a light period starting at 07:30 a.m.
2. Provide food and water ad libitum. Use 11-13 weeks-old rats weighing 300-400 g for the protocol.

2. Room set-up and preparation steps

1. Switch on the anesthesia station, set the heating mat to 37.5 °C, and cover it with a non-sterile drape.
2. Sterilize the surgical instruments required for the protocol: DeBakey atraumatic forceps (1), fine and sharp scissors (1), standard pattern forceps (2), needle holders (1), Vannas micro dissecting scissors (1), clamp (1).
3. Prepare catheters for the jugular vein and femoral artery.
   1. For the jugular vein catheter, mount Polyethylene (PE) tubing (PE50) on a 23 G needle with the tip cut off at the end.
   2. For the femoral artery catheter, prepare the one described in step 2.3.1 and attach a PE10 tubing to the end of the PE50 tubing.
4. Prepare the pressure transducer and catheters for the femoral artery and jugular vein by filling it with lactated Ringer's solution heparinized at 100 UI/mL. Place a 2 mL syringe containing lactated Ringer's solution heparinized at 100 UI/mL at the end of the catheters and the pressure transducer. Be careful not to get any bubbles, as these cause signal defects.
5. Switch on the software associated with the pressure transducer and calibrate it according to the supplier's instructions.

3. Preparing the rat for surgery

1. Anesthetize the rat with an induction box (parameters: sevoflurane 8%, air flow rate: 1 L/min). After checking the depth of anesthesia via pedal reflex, maintain anesthesia at sevoflurane 4%, with an airflow rate of 0.6 L/min.
   NOTE: From this step, the depth of anesthesia is assessed every 20 min via pedal reflex.
2. Weigh the rat, place it in the dorsal decubitus position on a heating mat, and depilate it at the inguinal and neck regions.
3. Disinfect depilated areas with alternating 10% and 4% povidone-iodine solutions (3 times each) using nonsterile gauze pads.
4. Apply a drop of ophthalmic ointment to the animal's eyes to prevent drying.
5. Place the rat on a heating mat on the surgical table (dorsal decubitus). Place the lubricated rectal probe to control the rat's temperature. Maintain anesthesia at sevoflurane 4%, with an airflow rate of 0.6 L/min.
6. Change gloves for sterile ones, place the sterile drape on the rat, and cut it at the inguinal and neck regions.

4. Jugular vein cannulation

1. Locate the jugular region in the lower right neck region, above the clavicle, by visible pulsation. Utilizing the DeBakey atraumatic forceps, gently grasp the skin, then make a precise incision using fine, sharp scissors.
2. Apply a drop of local anesthetic (lidocaine 2%) to the incised area. Gently slash tissue with standard pattern forceps.
3. Locate the jugular vein and gently liberate it from its muscular compartment with standard pattern forceps. Place a 4/0 silk suture thread and securely ligate the distal side (toward the head). Use the suture with the needle holders to tension the jugular vein.
4. Place a 4/0 suture on the proximal side (toward the heart) and prepare a surgeon knot without tightening it. Make a small incision in the jugular vein using Vannas micro dissecting scissors.
5. After carefully grasping the vein wall with small forceps, insert the PE50 catheter in the jugular vein by hand or with a standard pattern forceps. At this stage, use a sterile gauze pad to wipe eventual drops of blood, which may leak from the vein.
6. Advance the catheter slightly (0.5 cm) and verify its correct placement in the vein by drawing a small amount of blood and then re-injecting it (blood in the cannula indicates it is in the vein). Tighten the previously prepared knot to secure the catheter.
7. Leave the catheter in place, attached to a syringe pre-filled with lactated Ringer's solution, and cover the incised area with a moistened sterile gauze pad.

5. Femoral artery cannulation

1. Use DeBakey atraumatic forceps to grasp the skin of the Scarpa triangle of the left leg and make an incision with fine and sharp scissors. Apply a drop of local anesthetic (lidocaine 2%) to the incised area. Gently dilacerate tissue with standard pattern forceps.
2. Locate the femoral triad (artery, vein, and nerve). Pass one forceps under the triad and, using the second standard pattern forceps, very gently separate the artery from the nerve and femoral vein.
   NOTE: The artery is smaller, pink, and pulsating. This is a critical step; the vein and artery are fragile and can easily rupture.
3. Place a 4/0 suture and ligate the femoral artery distally (leg side). Take the suture with the needle holders to put in tension the femoral artery. Place a 4/0 suture on the proximal side (heart side) and prepare a knot without closing it. Clamp the artery (place the clamp upstream of the proximal side suture).
4. Incise the femoral artery transversely with Vannas micro dissecting scissors (a small volume of blood should flow from the artery). Using standard pattern forceps, gently grasp the artery wall and cannulate the femoral artery with the catheter (PE10 ended), holding it with forceps.
5. Gently unclamp to check for leaks and then check the pressure signal (validates that the catheter is in the femoral artery), and check that blood is not flowing back into the catheter (sign of leakage from the valves/pressure transducer).
6. If the signal is good (expected values: systolic blood pressure: 120 mmHg, diastolic blood pressure: 80 mmHg) and there are no signs of leakage, advance the catheter slightly (0.5 cm) and tighten the previously prepared surgeon knot.
7. Heparinize the animal at 100 UI/kg and apply a moistened sterile gauze pad over the incised area. After surgery, maintain anesthesia at sevoflurane 3% at an air flow rate of 0.6 L/min.

6. Hemorrhagic shock protocol (Figure 1)

1. Phase 1: Stabilization (5 min).
   1. After heparinization, wait 10 min for pressure values to stabilize. Make a 5 min recording for basal hemodynamic values.
2. Phase 2: Exsanguination (5 min):
   NOTE: This phase corresponds to the fixed-volume phase of the model.
   1. Draw 5 mL of blood from the femoral artery over 5 min (500 µL/30 s) using a 1 mL syringe (expected values: systolic blood pressure: 45 mmHg, diastolic blood pressure: 30 mmHg).
   2. Prepare a mixture of 50% lactated Ringer's solution with 50% collected blood at room temperature. Place the mixture in a 2 mL syringe and place it at the end of the jugular vein cannula.
3. Phase 3: Hemorrhagic shock (75 min)
   NOTE: This phase is the fixed-pressure phase of the model.
   1. Maintain mean arterial pressure at an average of 35 mmHg. When mean arterial pressure is over or equal to 38 mmHg, withdraw 200 µL of blood via the femoral artery (mean on n = 12 rats: 10.2 mL).
   2. If the mean arterial pressure drops below 32 mmHg, inject 200 µL of a 50% blood-50% lactated Ringer's solution mixture via the jugular vein (mean on n = 12 rats: 0.90 mL).
   3. Measure peripheral blood lactate on a blood drop from the end of the tail using the tip of a needle (26 G) after sterile preparation of the tail with a gauze pad at the end of the hemorrhagic shock phase.
4. Phase 4: Intravenous fluid resuscitation (20 min)
   1. Resuscitate with a lactated Ringer's solution at room temperature (RT) at 10 mL/kg over 20 min (flow rate of 10.5 mL/h for a 350 g rat) with a 20 mL syringe through the jugular catheter with a syringe pump.

7. End of surgery and recovery and post-surgical follow-up

1. Clamp the jugular vein and the femoral artery and remove the catheter. Ligate vessels. Check carefully that no blood is leaking out.
2. Add a drop of local anesthetic to the incised areas. Suture incised areas with subcutaneous and cutaneous stitches using 5-0 sterile suture. Disinfect with 10% povidone-iodine solution.
3. Subcutaneously inject buprenorphine (0.05 mg/kg, 0.3 mL/kg) using a 1 mL syringe with a 26 G needle.
4. Wait for the rat to wean from anesthesia while monitoring temperature. When it shows signs of waking up (vibrissae moving, paw movements), remove the rectal probe and place the rat in its cage on a heating mat. 10 min later, return the rat to his accommodation room.
5. Administer buprenorphine subcutaneously (0.05 mg/kg, 0.3 mL/kg) every 8 h.
6. Evaluate respiration rate, behavior, temperature, and blood lactate 16 h after hemorrhagic shock induction, as described in step 6.3.

Figure 1: Model of mixed rat hemorrhagic shock. Created with BioRender.com Please click here to view a larger version of this figure.

8. 24 h after hemorrhagic shock induction

1. Anesthetize the animal as described in step 3.1. Place the animal on the surgical table and insert the rectal probe as described in step 3.5. Prepare the catheter for carotid artery cannulation as described for the jugular vein in steps 2.3 and 2.4.
2. Use DeBakey atraumatic forceps to grasp the skin and make an incision in the middle of the neck with fine and sharp scissors. Apply a drop of local anesthetic (lidocaine 2%).
3. Gently dilacerate tissue with standard pattern forceps. Separate the salivary glands, then open the tracheal muscle to reveal the tracheal rings.
4. Take the left carotid artery and separate it from the nerve with standard pattern forceps (it is highly likely that the animal's breathing will accelerate). Ligate the carotid artery distally (head side) with 4/0 silk thread.
5. Place a suture thread on the proximal side (heart side) and prepare a surgeon knot without closing it. Clamp the artery (place the clamp upstream of the proximal side suture).
6. Incise the carotid artery with Vannas micro dissecting scissors (a small volume of blood should flow from the artery). Using standard pattern forceps, gently grasp the artery wall to enlarge the opening and cannulate the carotid artery with the catheter provided, holding it with forceps.
7. Unclamp and check the pressure signal to confirm catheter placement, and check that blood is not flowing back into the catheter (a sign of leakage from the valves/pressure transducer).
8. If the signal is good and there is no leakage, advance the catheter slightly (0.5cm) and tighten and secure the previously prepared surgeon knot.
9. Heparinize the animal at 100 UI/kg and apply a moistened sterile compress over the incision.
10. After surgery, maintain anesthesia using sevoflurane 3% at an airflow rate of 0.6 L/min.
11. Wait 10 min to record 24 h hemodynamic values (expected values: systolic blood pressure: 120 mmHg, diastolic blood pressure: 60 mmHg).
12. To assess plasmatic markers of organ damage, draw 1 mL of blood from the carotid artery and centrifuge at 1600 x g for 10 min. Aliquot and save plasma for further analysis.
13. Sacrifice the rat immediately after measuring hemodynamic values at 24 h and collect blood.
    NOTE: The method of euthanasia should be adapted to the specific parameters or samples that will be collected afterward. The sham group undergoes surgery only (sections 1-5, 7, and 8) without the procedure of hemorrhagic shock (section 6). Animals were randomly assigned by the experimenter between the sham and hemorrhagic shock groups. Only the experimenter was aware of the group allocation at the different stages of the experiment.