surgical procedure for intrapulmonary tracheal transplantation in mice

Exploring Obliterative Airway Disease in Lung Transplants

Lung transplantation has been established as an effective treatment for patients with end-stage respiratory diseases. However, the median survival rate for human lung transplant recipients is only approximately 6 years, with the development of obliterative bronchiolitis (OB), a type of obstructive airway disease (OAD), being a major cause of death after the first year post transplantation1.
Several animal models have been utilized to investigate the mechanism underlying OAD. One such model is the heterotopic tracheal transplantation (HTT) model2. In this model, tracheal grafts are implanted into the recipient&#39;s subcutaneous tissue or omentum. Ischemia-induced loss of tracheal graft epithelial cells occurs, followed by alloreactive lymphocyte infiltration and apoptosis of donor epithelial cells. Fibroblasts and myofibroblasts migrate around the trachea, producing an extracellular matrix. Finally, complete fibrous obliteration of the airway lumen occurs. The HTT model is technically simple, provides an in vivo environment, and offers high reproducibility.
Another model for studying OAD is the rat orthotopic tracheal transplantation (OTT) model, where tracheal grafts are interposed into the recipient&#39;s trachea to maintain physiological ventilation3. In this model, ischemia-induced depletion of donor epithelial cells results in their replacement by recipient epithelial cells within the trachea, forming an unobstructed airway accompanied by moderate fibrosis. Although these models have contributed to the understanding of airway obliteration after lung transplantation, they have limitations in terms of recapitulation of the lung parenchymal microenvironment.
Our research group introduced the rat intrapulmonary tracheal transplantation (IPTT) model, where tracheal grafts are implanted into the recipient lung4 (Figure 1). The IPTT model exhibits fibrous obliteration of the airway lumen occurring within the lung microenvironment. Furthermore, it has been successfully applied to mice that are technically more challenging than rat IPTT5,6,7,8,9,10. This adaptation of the murine IPTT model enabled us to delve deeper into the intricate details of the lung immunological environment of OAD after lung transplantation using transgenic mice.
The IPTT model possesses some unique features. One is neoangiogenesis, which is facilitated by pulmonary circulation and plays a crucial role in airway obliteration4,10. Additionally, the IPTT model exhibits lymphoid aggregates, some of which have high endothelial venules expressing peripheral node addressin, indicating that they are tertiary lymphoid organs (TLOs)7,8. TLOs resemble lymph nodes and consist of T cells, B cells, and frequently, a germinal center accompanied by follicular dendritic cells11,12. TLOs have been reported in various chronic inflammatory diseases, including airway obliteration, making the IPTT model suitable for investigating the role of TLOs in airway obliteration7,8,11,12,13. This paper presents the methodology of the murine IPTT model, with the goal of familiarizing researchers with this model and facilitate further investigations into airway obliteration following lung transplantation.

Author Spotlight: Investigating the Key Factors of Obliterative Bronchiolitis After Lung Transplantation

Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation

Our research is examining chronic lung allograft dysfunction after lung transplantation. When you transplant a lung, over half the patients will develop fibrous obliteration or abnormalities of the airway. Understanding this is really the key to understanding long-term survival after lung transplantation.The importance of this model of intrapulmonary tracheal transplant is that the transplanted trachea is placed into the environment of the lung itself, and therefore you can look at an allograft versus an Isograft in terms of its response after a transplant. Most fascinating is the fact that an Isograft, even though it's had the same ischemia and the same injury, completely recovers and has normal anatomy, but an allograft develops fibrous obliteration, and understanding why that happens in an allograft is the key to understanding the underlying mechanisms of fibrosis in lung transplantation.

Watch this Scientific Journal Video about Surgical Procedure for Intrapulmonary Tracheal Transplantation in Mice at JoVE.com

Surgical Procedure for Intrapulmonary Tracheal Transplantation in Mice

To begin, record the weight of each mouse, then position the euthanized mouse in a supine orientation and secure its limbs with tape. Use 70%isopropyl alcohol to sterilize the surgical area. Next, create a midline incision on the skin starting from the mid-abdomen to the anterior cervical region.To access the trachea, carefully retract the fat pads, laterally move the strap muscles and separate the trachea from the surrounding connective tissue. Using forceps, create a space between the trachea and the esophagus. Then lift the xiphoid and cut the diaphragm.Insert a hemostat to raise the sternum for an unobstructed path from the sternum to the neck region. Clamp the rib cage on both sides and cut through the sternum extending through the neck muscles. Now remove the thymus and any fat or muscle obstructing the trachea to expose the tracheal bifurcation.Cut the main bronchi and carefully separate the airway from the esophagus. Afterward, cut the larynx and remove it. Spray the dissected trachea with a sterile saline solution or a preservation solution.Use sterile gauze soaked in sterile saline or preservation solution and place it on ice to maintain viability. After lightly anesthetizing the mouse and an induction chamber, intraperitoneally inject a cocktail consisting of xylazine and ketamine. Return the mouse to the induction chamber with 2 to 3%isoflurane maintained.Then shave the fur at the surgical site. Confirm the absence of reflex response to a toe pinch before orotracheal intubation. Using a 20 gauge intravenous catheter, intubate the mouse orotracheally and connect it to a ventilator with 50%oxygen and 2%isoflurane.Now, activate a heating pad and position the mouse in a right lateral position on the pad with the head away from the surgeon and the tail towards the surgeon. Then secure the limbs with tape. Apply veterinary ointment to the eyes to prevent dryness while under anesthesia.Scrub the surgical area first with 7.5 povidone iodine, then 70%isopropyl alcohol, and finally, with 10%povidone iodine. During this time, load the donor trachea into a 16 gauge intravenous catheter. After making an incision in the recipient's skin, cauterize the muscle and connective tissue.Using two retractors, open the fifth or sixth intercostal space and hold the rib cage open. Then use cotton swabs and scissors to dissect the inferior pulmonary ligament. Simulate the creation of the pathway for the donor trachea.To secure the ventilator outflow tube, partially occlude it with a three-way stopcock to facilitate inflation of the left lung. Puncture the left lung with a 20 gauge needle to create a pathway. Then insert the 16 gauge intravenous catheter into the left lung and extrude the donor trachea into it.After insertion of the tracheal allograft, release the three-way stopcock to allow unobstructed expiratory flow through the outflow tube. To close the pleural injection site, position the clip precisely onto the puncture site with its edge aligned to the contour of the lung's edge. Then fill the thoracic cavity with saline solution and absorb the saline with gauze.Reinflate the left lung and close the ribs using a running suture. Close the muscle and skin with interrupted sutures. Now, turn off isoflurane and administer meloxicam analgesic subcutaneously at the end of the surgery.Observe the recipient mouse until it is awake. Then remove the tracheal tube and put the recipient mouse in a cage. A tracheal allograft exhibited complete obstruction with fibroblastic tissue, and the epithelial cells were visibly destroyed.Conversely, a tracheal isograft remained patent, and the epithelial cells were preserved. Lymphoid aggregates were observed in the lung with the transplanted tracheal allograft.

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152 Görüntüleme.  University Health Network. Başlamak için, her farenin ağırlığını kaydedin, ardından ötenazi fareyi sırtüstü yönde konumlandırın ve uzuvlarını bantla sabitleyin. Cerrahi bölgeyi sterilize etmek için %70 izopropil alkol kullanın. Daha sonra, orta karın bölgesinden başlayarak ön servikal bölgeye kadar cilt üzerinde bir orta hat kesisi oluşturun.Trakeaya erişmek için, yağ yastıkçıklarını dikkatlice geri çekin, kayış kaslarını yanal olarak hareket ettirin ve trakeayı çevrele...