Die Transplantation autologer Lymphknotenfragmente als Therapiemöglichkeit des sekundären Lymphödems

An effective transport of body fluids is only guaranteed by a functional lymphatic system. Within the context of an axillary dissection during breast cancer treatment this balance can be disturbed, resulting in a secondary lymphedema in about 30% of the patients. The therapy of secondary lymphedema is only symptomatical so far. As a possible surgical therapy, fragments of autologous lymph nodes had been transplanted to prevent lymphedema in rats. VEGF-C showed a beneficial effect in this context. The VEGF-C application pattern with regards to the parameters “time point”, “location” and “dosage” remained unexplored. The aim of this study was to verify these parameters and determine their effects on the regeneration and reconnection of the transplants by using this rat model. In addition, the effects of an extended recovery period (8 weeks) on the regeneration of the transplants were also investigated. During the studies the surgical technique of this rat model caused difficulties regarding the transplant removal. The transplants got attached to the node of the suture, thus the sampling was only manageable with a loss of transplant substance. Therefore, the secondary aim consisted in altering the technique so that a removal without any loss of the substance is possible. Ninety-six adult, healthy female Lewis rats (approximately 200g) underwent surgery, in which all right popliteal and inguinal lymph nodes were removed. Three inguinal lymph nodes of each rat were fragmented and transplanted back into the subcutaneous fat tissue of their right groin by using the modified surgical procedure in which an attachment of the transplants to the node was avoided. The rats in group A (10 rats) were injected with 6.67µg VEGF-C per rat intradermally into the right abdominal wall close to the transplantation area on day 1, 2, 3 post OP. All animals in group B (19 rats) were given 6.67µg VEGF-C per rat intradermally in the medial side of the right thigh on day 1, 2, 3 post OP. In group C all rats (20) were injected with the same dosage of VEGF-C and at the same location as group B, but on day 14, 15, 16 post OP. The rats in group D (20 rats) were injected 13.34µg VEGF-C per rat on the same time points and location as group B. In group E (20 rats) there was no further intervention after surgery, likewise in the pilot group (7 rats). After 4 weeks (group A-E) or 8 weeks (pilot group) the lymphatic drainage and the connection of lymphatic vessels with the transplants were tested. Therefore, Patent Blue was injected intradermally into the medial side of the right thigh. The distribution of the dye was examined and graded as “rat with transplant reconnection” or “rat without transplant reconnection”. Afterwards the transplants were prepared for HE staining (pilot group, group A-E) and immunohistochemistry (group A-E) to detect T-lymphocytes, B-lymphocytes, HEVs and lymphatic endothelial cells. The rats were graded as “rat with regenerated transplants” or “rat without regenerated transplants” due to the cell distribution detected by fluorescent immunohistochemistry. Pilot Study: After a recovery period of 8 weeks 4 out of 7 (57%) rats showed possibly regenerated lymph nodes. In group E 14 out of 20 (70%) of the animals revealed regenerated lymph nodes after a recovery period of 4 weeks. Surgery modification: The removed transplants showed no attachment to the node of the suture and in only 2 of the rats (2%) were the transplants found in the close surrounding of the transplantation area; they did not remain fixed to the non-absorbable suture. Transplant reconnection with the lymphatic system: In group A 5 out of 10 rats (50%) showed a reconnection of the transplants with the lymphatic system. In group B 10 out of 19 rats (53%) and in group C 7 out of 20 rats (35%) had a reconnection of the transplants. In group D there were 17 out of 20 rats (85%) with a detectable transplant reconnection. The control group (group E) showed a reconnection in only 3 out of 20 rats (15%). Transplant regeneration: In group A 8 out of 10 rats (80%) and in group B 17 out of 19 rats (89%) showed regenerated lymph nodes. In group C there were 17 out of 20 rats (85%) with a successful regeneration of the transplants and in group D there were 18 out of 19 rats (95%). The control group (group E) showed 14 out of 20 rats (70%) with regenerated lymph nodes. This work shows that an avascular transplantation of autologous lymph node fragments is possible and that an extended recovery period does not result in better regeneration rates. A change in the surgical procedure leads to an easier sampling while avoiding loss of substance. Furthermore, VEGF-C enhances the regeneration of the transplants and the reconnection of lymph node fragments with the lymphatic system. An early time point on days 1, 2 and 3 post OP and the intradermal injection into the medial side of the thigh especially improve the reconnection of the transplants with the lymphatic system. The modifications in both of the parameters do not affect the regeneration of the transplants statistically. Only the dosage shows an effect on the transplant reconnection as well as the transplant regeneration. A dosage of 13.34 µg VEGF-C revealed a statistical tendency to improve regeneration; it also enhances the reconnection extremely significantly. This study confirms that the avascular transplantation of autologous lymph node fragments is a possible therapy for secondary lymphedema, especially with respect to the prevention after breast cancer treatment.