F. Fändrich¹, J. Schröder¹, T. Jahnke¹, A.M. Waaga³, M.R. Pawaresch², H.H. Wacker²

¹Dept. of General & Thoracic Surgery and ²Institute of Pathology, University of Kiel, Germany.
³Brigham and Women's Hospital, Harvard Medical School, Boston, MA, U.S.A.

Received 12/2/96; Accepted 12/18/96; On-line 01/01/97

MATERIALS AND METHODS

In this study we established a semiallogeneic parent to F1 hybrid graft-versus-host model by use of male parental DA (RT1.^aav1) inbred donor rats and female (LEW(RT1^l) x DA) F1-hybrid recipients. Heterotopic small bowel transplantation (HSBTx) was performed according to a modified technique of Monchik and Russell as previously published (10). Briefly, total small bowel was harvested with aortic cuff and portal vein, followed by perfusion with cold saline. For the revascularization, the graft aortic cuff was anastomosed to the infrarenal abdominal aorta. The graft portal vein was anastomosed to the infrarenal vena cava. The proximal gut lumen was closed. The distal end was inserted in an end-to-side fashion into the recipient's distal ileum. Animals (age between 16 and 20 weeks) were purchased at the "Zentralinstitut für Tierzucht Hanover, FRG" and weighed between 250-300g. Animal use and care respected the guidelines established by the German Council on Animal Care.

Animal Groups

Experiments included the following three groups (n=30):

Group 1: HSBTx of DA » F1, DOS 5mg/kg rat, from day 0 to day 14

Group 2: HSBTx DA » F1, no treatment

Group 3: HSBTx F1 » F1, no treatment

Animal treatment

Lyophilized 15-deoxyspergualin was resuspended in 1 ml of saline (0.9%) and injected intramuscularly at a dose of 5mg/kg body weight from day 0 to day 14 following HSBTx.

Immunohistology

Small bowel grafts and mesenteric lymph nodes of donor and recipient together with spleen, donor and recipient mesenteric lymph nodes and small bowel specimens were harvested on day 3, 7, 14 and 21 following SBTX. The tissues were sliced into small pieces, either snap-frozen in liquid nitrogen and stored at -80°C or subjected to 10% buffered formaldehyde, embedded in paraffin, cut at 4µm and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed using an indirect APAAP-staining technique (11). The panel of monoclonal antibodies (mAbs) used to characterize different lymphocyte populations included: Ki-M2R (macrophages), Ki-B1R (B-cells), Ki-T1R (pan-T cells), Ki-M4R (follicular dendritic cells) (12), and Ki-M9R (sinus lining cells) (1). After staining, the number of positive cells was determined and expressed as a percentage of the total cell count seen in 4 high-power fields per specimen. H&E-stained sections allowed the diagnosis of GvHD in recipient small bowel tissues as confirmed by the accepted histological parameters as described by Grant (9).

Flow cytometric analysis

At postoperative day 3, 7, 14, and 21 donor and recipient mesenteric lymph nodes and spleen were harvested from recipient animals to perform fluorescence activated cell sorting (FACS) analysis. For assessment of T and B lymphocytes and macrophages, monoclonal antibodies were used as follows: MRC OX19 (mouse-anti-rat pan T cells); MRC OX12 (mouse-anti-rat B cells) and MRC OX41 (mouse-anti-rat macrophages) purchased at Camon, Wiesbaden-Germany. Peroxidase-conjugated rabbit anti-mouse (Dakopatts, Glostrup, Denmark) and peroxidase conjugated affinity pure F(ab')₂ FITC-labeled goat anti-rabbit IgG (Dianova, Hamburg-Germany) monoclonal antibodies were used as secondary antibodies.

Statistics

Survival curves were generated according to the method described by Kaplan-Meier. The two-sided log-rank test was used to assess significant differences in animal survival.