Vascular Thymus and Hind Limb Allotransplantation Model

Fig. 23.1

Schematic presentation of vascularized thymus transplantation. (a) Donor. The right lobe of the thymus on its vascular pedicle, including the subclavian artery and superior caval vein, is harvested. (b) Recipient. Thymus is placed in the right side of the recipient’s neck and is anastomosed to the common carotid artery and to the jugular vein

The right lobe of the thymus, after all thymus-associated lymph nodes are removed, is separated from the left lobe. The subclavian artery is clamped just distal to the bifurcation and sharply transected. The superior caval vein also sharply cut horizontally just proximal to the right atrium. The right lobe of the thymus on its vascular pedicle, including both the subclavian artery and superior caval vein, is harvested and placed in the right side of the recipient’s neck prepared previously. The subclavian artery of the harvested thymus is anastomosed to the recipient’s common carotid artery, and superior caval vein to the jugular vein, using conventional end-to-end microsurgical with 9/0 sutures, respectively. The transplanted right lobe of the thymus is positioned within the neck cavity created by removal of the sternal head of sternocleidomastoid muscle. The skin is closed with 5/0 Vicryl suture.

Immediately following transplantation, perfusion of the transplanted thymus is evaluated by two different microsurgical patency tests at 5 and 30 min after anastomosis: (a) the milking, and (b) the hook patency tests. The flow of the anastomosed subclavian artery and superior caval vein are evaluated with a Model T-206 flowmeter (Transonic). Histologic H&E sectioning and examination is done at the end of the planned experimental study.

Preparation of the Creamaster Muscle for In Vivo Microscopic Assessment of Microcirculation (Lewis-Brown Norway to Lewis rat Hindlimb-Thymus)

At the end of each follow-up period, the creamaster muscle is withdrawn from the medial border of the hindlimb, opened in the front wall with thermal cautery, spread over the cover glass of tissue bath, and fixed with 5/0 silk sutures. The muscle is kept moist and covered with oxygen-impermeable plastic film (plastic Wrap presoaked for 24 h in distilled water). The temperature of the system is maintained between 35 and 37 °C.

Protocol for Direct In Vivo Observation of Microcirculation

The Zeiss 20 T epifluorescence microscope, equipped with a Hitachi KP-C503 color video camera, a Sony Trinitron 19-in. monitor, and Panasonic AG-1730 video recorder, is used. The creamaster muscle on the tissue bath is transilluminated from below using a fiber-optic tungsten lamp. The final magnification on the screen is 1,800×. The following parameters are measured (in microns).

White Blood Cell Count in Postcapillary Venules

Video scans of three preselected postcapillary venules (30–40 μm in diameter) are taken as follows. One postcapillary venule is chosen in each of the regions of the creamaster flap: proximal, middle, and distal. A 50- μm-long segment of the postcapillary venule is choosen observation and recording. For each venule, the number of rolling, adhering, and transmigrating leukocytes and lymphocytes is counted during a 2-min period a hand counter.

Endothelial Edema Index

The ratio of the outer venular diameter to the inner diameter is measured in three preselected postcapillary venules in the proximal, middle, and distal parts of the muscle flap using a video image marker-measurement system (VIA-150, Boeckeller, Tucson, Ariz.). This ratio reflected changes in the thickness of the postcapillary venular wall caused by endothelial edema, which is related to the deposition of blood morphotic elements within the vessel lumen.

All microcirculatory images are video-taped, allowing for repeated observations, including slow-motion replay.