V. R. Rendell3, J. Lin2, M. L. Markert2, T. V. Brennan1 1Duke University Medical Center,Surgery,Durham, NC, USA 2Duke University Medical Center,Pediatrics,Durham, NC, USA 3Duke University Medical Center,Medicine,Durham, NC, USA
Introduction: Rodent thymectomy enabled defining the role of the thymus in lymphocyte differentiation, self-tolerance, and immunotolerance in the setting of allograft transplantation and tumor metastasis. The technique facilitates studies involving T-cell depletion or the adoptive transfer of defined T cell populations without the re-emergence of native naïve T cells. Thymectomy in adult rats has proven to be more challenging than neonatal rat thymectomy due to increased rates of bleeding, tracheal injury and pneumothorax. The objective of the present study was to develop a technique for adult rat thymectomy with low complication rates and reliable complete removal of the thymus.
Methods: The protocol was evaluated for 26 rats. An endotracheal intubation cannula was fashioned from an angiocatheter needle. Rat intubation was accomplished using trans-cutaneous tracheal illumination to visualize the aperture of the larynx prior to insertion of the prepared cannula within the angiocatheter tubing. A minimally invasive 2 cm skin incision was followed by a 1.5 cm median sternotomy and division of the pre-tracheal muscles. The inferior thyroid lobes were carefully dissected away from the pleural lining. After complete removal of the thymus, a three layer closure with surgical glue application was performed. Removed tissue was stained by H&E and with cytokeratin staining. Peripheral blood from the rats was analyzed for persistent depletion of naïve T cells post-operatively by staining with naïve T cell markers and dividing the percent of each cell type present by the absolute lymphocyte count.
Results: The mean intubation time was 45sec +/- 5 sec, and her mean operation time was 15 ± 3 min. There was an associated 96% immediate post-operative survival rate. There was no intraoperative mortality. Twenty-four rats recovered fully. One rat survived after re-exploration of the chest. All removed thymus tissue was submitted to path. Immunohistochemistry cytokeratin would help differentiate thymic tissue from lymph nodes. All thymectomies performed were confirmed to be complete. Rats who underwent thymectomy had minimal new naïve type T cells post-operatively. The thymectomized rats maintain overall T cell counts but have diminished naïve T cells.
Conclusion: This method is appropriate when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats. 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.