Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Summary

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Abstract

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of naïve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Introduction

Since the early 1960s, the thymus has been recognized for its critical role in the development of central immunological tolerance. Rodent thymectomy has proven to be an essential procedure in defining the role of the thymus in lymphocyte differentiation, self-tolerance, and immunotolerance in the setting of allograft transplantation and tumor metastasis. Removal of the rat thymus allows for studies involving T-cell depletion or the adoptive transfer of defined T cell populations without the re-emergence of native naïve T cells.

Thymectomies in neonatal rodents can be accomplished using a suction technique with reliable outcomes¹

Protocol

NOTE: All experimental procedures involving the use of rats were done in accordance with protocols approved by the Animal Care and Use Committee of Duke University.

1. Preparation of the Tracheal Intubation Cannula

1. Cut off the needle end of a 2 inch 14 G angiocathether needle with cutting pliers.
2. Pinch the lumen of the needle closed with needle-nosed pliers.
3. Cut the pinched section down to the edge of the open-lumen portion with cutting pliers, and then cut the edges on both sides of the remaining end at 30 - 45° angle to the needle edge.
4. Place the bottom 3 - 4 mm of the cannula into the open a....

Results

This procedure was performed on adult Lewis rats (n = 26). The mean operation time was 15 ± 3 min. There was no intraoperative mortality. The mean intubation time – from placement of rat on the intubation apparatus to establishment of ventilation – was 45 ± 5 sec. 24 rats had a full recovery from the operation with no evidence of respiratory difficulties or hemorrhage through post-operative day (POD) 14. One rat developed respiratory difficulty on POD 4 and underwent re-operation to explore the mediastinum. The rat was f.......

Discussion

The present protocol for complete thymectomy provides a minimally invasive approach with a three layer incision closure with surgical glue application designed to minimize complications. Complete removal of the thymus was demonstrated by the loss of naïve T cells and by IHC staining of mediastinal lymphoid tissue for cytokeratin.

The procedure of adult rat thymectomy has been complicated by mortality rates ranging from 1.5 - 20% due to peri-operative complications, most of which are the a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 inch 14 G angiocatheter
Operating microscope	Zeiss
Warming pad
Heart rate and blood oxygenation monitor for rodents with foot sensors	Harvard Apparatus	ST1 72-8010, ST1 72-8098 (Rat foot sensor)
Intubation apparatus (plastic with metal bar at the top)			See Figure 2
Small animal anesthesia system with induction box, isoflurane tank and O2 tank	Harvard Apparatus	ST1 72-6420
Small animal ventilator with tubing	CWE	12-02000 (ventilator) and 12-04000 (external valve assembly for mice/rats)
High-intensity fiber optic Illuminator	Dolan Jenner	EEG 2823M
Student standard pattern forceps	Fine Science Tools	91100-16
Fine straight scissors	Fine Science Tools	14060-09
Blunt-tipped Shea scissors	Fine Science Tools	14105-12
Small Alm retractor (for sternum)	Fine Science Tools	17008-07
Blunt Graefe forceps	Fine Science Tools	11050-10
Fine Dumont forceps	Fine Science Tools	11254-20
5-0 Maxon sutures	Ethicon
4-0 Silk sutures (with cutting needle)	Ethicon
6-0 Nylon suture	Ethicon
Cyanoacrylate glue (Endermil)
Lubrication gel	Akorn Animal Health	NDC 17478-162-35