A Model of Acute Lung Injury Following Visceral Ischemia-Reperfusion by Supra-Coeliac Aortic Cross Clamping in Rats

We present a reproducible rat surgical model of acute lung injury after supra-coeliac aortic cross clamping, enabling detailed study of ischemia perfusion mechanism in preliminary injury. Our protocol is feasible, reproducible, low-cost, and easy to learn, enabling rapid implementation, biomarkers exploration, and testing of diverse pharmacological interventions. Our surgical model of acute lung injury will enable us to investigate various pharmacological strategies aimed at reducing the severity of pulmonary damage following aortic cross clamping.

To begin, place an anesthetized rat in dorsal recumbency and immobilize the front and hind limbs with adhesive tape while maintaining physiological joint motion ranges. Transfer the rat onto a warming pad set to 37 degrees Celsius and monitor rectal temperature. Apply ophthalmic eye ointment to the eyes to prevent dryness.

After shaving the abdominal fur, thoroughly disinfect the area using povidone iodine solution and alcohol. To perform the surgery, make a skin incision at the center of the abdomen to expose the linea alba. Incise the abdominal muscle centered on the linea alba to prevent bleeding from surrounding muscle tissues.

Maintain the abdominal wall on both sides using self-retaining retractors. Examine the cavity with cotton-tipped buds for any anomalies. Then, gently mobilize the viscera, stomach, spleen, and liver to the right side of the abdomen to expose the supra-coeliac aorta.

Keep all viscera inside the abdominal cavity using a compress folded lengthwise and soaked in warm saline solution at 37 degrees Celsius. Then, cover all exposed viscera with a compress soaked in warm saline solution at 37 degrees Celsius to prevent hypothermia and dehydration. Identify and encircle the supra-coeliac aorta with a 5-0 suture using dissecting and ligature forceps.

Identify the subrenal inferior vena cava through the transparent retroperitoneum. For the intravenous injection, use a 30-gauge needle and apply a small piece of hemostatic compress at the injection site to achieve hemostasis. Now, clamp the coeliac aorta with an atraumatic microvascular clamp for 40 minutes.

Verify the clamp's effectiveness by confirming the disappearance of aortic pulsations and discoloration of the viscera and limbs multiple times during clamping. Close the abdomen partially using three simple 5-0 braided absorbable sutures to reduce water and heat loss. Then, cover the incision with a sterile compress soaked in warm saline for the duration of ischemia.

At the end of the ischemic period, reopen the abdomen and expose the aorta. Remove the vascular clamp and verify resumption of aortic pulsations and recoloration of the viscera and limbs. This marks the beginning of the reperfusion period.

Return the viscera to their original position. Then, close the abdominal wall with a 5-0 braided absorbable suture on the linea alba. Use a 5-0 monofilament absorbable suture on the skin.

Place the rat in the left lateral decubitus position to prevent compression of the inferior vena cava during hypovolemia. Then, transfer it into an individual cage under a heat lamp until it awakens, which occurs approximately 10 minutes later. For blood sampling, directly puncture the inferior vena cava below the renal veins using a five milliliter syringe equipped with a 23-gauge needle.

After the animal has been euthanized, harvest the left kidney, then harvest the left hepatic lobe and the distal small intestine, one centimeter before the cecum, through the open abdomen. Quickly perform a median sternotomy to expose and identify the heart and lungs. Free the left lung from its ligament and remove it entirely for histological analysis.

Then, free the right multi-lobed lung from its ligaments and isolate the anterior lobe for nucleic acid and protein analysis. Extract the heart as a whole block, including sections of the aorta, pulmonary artery, and both vena cavi. Blood gas analysis following supra-coeliac aortic cross clamping showed reduced pH, decreased partial oxygen pressure, increased partial carbon dioxide pressure, and lower bicarbonate levels compared to normal ranges.

The histopathologic lung injury score increased markedly in the supra-coeliac aortic cross clamping group. Polymorphic nuclear neutrophils per square millimeter were significantly elevated after supra-coeliac aortic cross clamping. Intravascular leukocyte counts were significantly higher in the aortic cross clamping group compared to the sham group.

Pulmonary inflammation markers were also significantly elevated after supra-coeliac aortic cross clamping. Additionally, pulmonary edema as measured by gravimetric analysis was found to be increased after aortic clamping.