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Regional Inflammation Following Lymph Node Transfer Improves Spontaneous Lymphatic Reconnection and Functional Drainage
Walter J. Joseph, BS, Seth Z. Aschen, BS, Gina Farias-Eisner, BS, Daniel A. Cuzzone, MD, Swapna Ghanta, MD, Nicholas J. Albano, BS, Ira L. Savetsky, MD, Jason C. Gardenier, MD, Babak J. Mehrara, MD.
MSKCC, New York, NY, USA.
Lymph node (LN) transplantation has been clinically shown to improve lymphatic function and decrease lymphedema in selected cases. However, the results of these reports have been inconsistent as a consequence of inadequate lymphatic regeneration in some patients. Therefore, developing novel strategies that improve lymphatic regeneration after LN transplantation is clinically relevant and important. We have previously shown that lymphatic vessels spontaneously reconnect and restore lymphatic function after LN transplantation in a mouse model. In addition, we have previously shown that sterile inflammation massively increases lymphangiogenesis. Therefore, the purpose of the current study was to determine if sterile inflammation before or after LN transplantation can be used as a means to improve spontaneous lymphatic reconnection and lymphatic function.
To test the hypothesis that sterile inflammation can improve lymphatic reconnection after LN transplantation, we performed LN transplants in adult male mice using lymphatic reporter mice (express B-gal in lymphatic vessels) as recipients and wild-type mice as LN donors. The axillary contents of recipient mice including LNs and perinodal fat were replaced with tissues from the donor mice. Animals were divided into 2 experimental groups. In group I, we induced sterile inflammation in draining LNs by injecting Freund’s Complete Adjuvant (CFA)/OVA in the distal extremity of the donor mouse 14 days prior to transplantation. In group 2, we performed LN transplantation and then injected CFA/OVA in the distal extremity of the recipient mouse 14 days following transplantation. A third group of animals served as inflammation controls and were not treated with CFA/OVA before or after surgery. Four weeks after surgery, we compared lymphatic vessel regeneration, VEGF-C expression, and lymphatic function with sham operated control animals that had undergone axillary incision without lymph node removal.
Sterile inflammation prior to LN transplantation resulted in signification donor LN enlargement and lymphangiogenesis. However, unexpectedly we found that inflammation prior to LN transplantation was associated with markedly impaired lymphatic regeneration as compared with animals that did not have inflammation or those in which inflammation was induced after transplantation. For example, analysis of lymphatic function using Tc99 lymphoscintigraphy demonstrated that induction of inflammation after LN transplantation resulted in a 1.68-fold increase in Tc99 uptake in the transplanted node as compared with pre-inflammation transplanted lymph nodes (p=0.0002). Lymphatic vessel staining corroborated these findings demonstrating a massive ingrowth of lymphatics into the post-transfer inflammation LNs as compared to pre-transfer inflammation group. These findings also corresponded to increased lymphangiogenic cytokine expression in the post-transfer inflammation group. Post-transplant inflammation animals had virtually normal lymphatic function as compared with sham-operated controls.
This study demonstrates that sterile inflammation after LN transplantation can be used as a means of augmenting lymphatic regeneration and function. Interestingly, we found that inflammation prior to transplantation markedly impairs lymphatic regeneration (despite increasing the size and level of lymphangiogenesis in the transplanted LN). Our findings also suggest that post-transplantation sterile inflammation increases the endogenous expression of lymphangiogenic cytokines and that this effect increases lymphatic vessel ingrowth in the transplanted LN.
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