Abstract
Vascularized lymph node transfer (VLNT) is a promising treatment modality for lymphedema; however, how lymphatic tissue responds to ischemia has not been well defined. This study investigates the cellular changes that occur in lymph nodes in response to ischemia and reperfusion. Lymph node containing superficial epigastric artery-based groin flaps were isolated in Prox-1 EGFP rats which permits real time identification of lymphatic tissue by green fluorescence during flap dissection. Flaps were subjected to ischemia for either 1, 2, 4, or 8 hours, by temporarily occluding the vascular pedicle. Flaps were harvested after 0 hours, 24 hours, or 5 days of reperfusion. Using EGFP signal guidance, lymph nodes were isolated from the flaps and tissue morphology, cell apoptosis, and inflammatory cytokines were quantified and analyzed via histology, immunostaining, and rtPCR. There was a significant increase in collagen deposition and tissue fibrosis in lymph nodes after 4 and 8 hours of ischemia compared to 1 and 2 hours, as assessed by picrosirius red staining. Cell apoptosis significantly increased after 4 hours of ischemia in all harvest times. In tissue subject to 4 hours of ischemia, longer reperfusion periods were associated with increased rates of CD3+ and CD45+ cell apoptosis. rtPCR analysis demonstrated significantly increased expression of CXCL1/GRO-α with 2 hours of ischemia and increased PECAM-1 and TNF-α expression with 1 hour of ischemia. Significant cell death and changes in tissue morphology do not occur until after 4 hours of ischemia; however, analysis of inflammatory biomarkers suggests that ischemia reperfusion injury can occur with as little as 2 hours of ischemia.
Highlights
Lymphedema is a chronic progressive disease characterized by tissue swelling and lymph fluid stasis that leads to tissue fibrosis, limb disfigurement, and loss of function
Since we recently developed and characterized a transgenic rat which facilitates in situ real time visualization of lymphatic tissue (Prox1-EGFP), this has enabled us to precisely identify and isolate lymph nodes from surrounding tissue and analyze the cellular changes that occurred within lymph nodes [23]
Lymph node flaps based on the superficial inferior epigastric artery and vein (SIEA/V) as described in the methods section were elevated and rendered ischemic by atraumatic occlusion of the femoral artery and vein proximal and distal to the pedicle for 1, 2, 4, or 8 hours (n = 12 rats per ischemic period; n = 48 total), after which the incision was reopened, and the microvascular clamps were removed to allow vascular reperfusion (Fig 2)
Summary
Lymphedema is a chronic progressive disease characterized by tissue swelling and lymph fluid stasis that leads to tissue fibrosis, limb disfigurement, and loss of function. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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