Prevention of postsurgical lymphedema via immediate delivery of sustained-release 9-cis retinoic acid to the lymphedenectomy site

Abstract

Background and objectives: Previously, we have shown that 9-cis retinoic acid (9-cis RA) stimulates lymphangiogenesis and limits postsurgical lymphedema in animal models when administered via daily intraperitoneal injections. In this study, we investigate whether a single-use depot 9-cis RA drug delivery system (DDS) implanted at the site of lymphatic injury can mitigate the development of lymphedema in a clinically relevant mouse limb model.

Methods: Hind limb lymphedema was induced via surgical lymphadenectomy and irradiation. Animals were divided into two treatment groups: (1) 9-cis RA DDS, (2) placebo DDS. Outcomes measured included paw thickness, lymphatic clearance and density, epidermal thickness, and collagen deposition.

Results: Compared with control animals, 9-cis RA-treated animals had significantly less paw swelling from postoperative week 3 (P = .04) until the final timepoint at week 6 (P = .0007). Moreover, 9-cis RA-treated animals had significantly faster lymphatic clearance (P < .05), increased lymphatic density (P = .04), reduced lymphatic vessel size (P = .02), reduced epidermal hyperplasia (P = .04), and reduced collagen staining (P = .10).

Conclusions: Animals receiving 9-cis RA sustained-release implants at the time of surgery had improved lymphatic function and structure, indicating reduced lymphedema progression. Thus, we demonstrate that 9-cis RA contained within a single-use depot DDS has favorable properties in limiting pathologic responses to lymphatic injury and may be an effective strategy against secondary lymphedema.

Keywords: 9-cis retinoic acid; depot; drug delivery system; drug pellet; lymphangiogenesis; lymphedema; postsurgical lymphedema; retinoic acid; secondary lymphedema; sustained release.

Figure 1. Timeline of the experimental methodology

Starting from day 0 (day of surgery), paw measurements were obtained weekly for a total of 6 weeks. At the final timepoint, animals underwent lymphatic clearance analysis before being euthanized for tissue harvesting.

Figure 2. Schematic of the hind limb lymphadenectomy model

(A) To induce hind limb lymphedema in mice, the inguinal fat pad (in yellow, indicated by the open arrow) and the popliteal lymph node (in green, indicated by the closed arrow) were surgically dissected. A circumferential skin incision was then made around the thigh along the dotted line, followed by hind limb irradiation. The red square indicates the field of view for images C–F. (B) Implantable pellets measured 3 mm x 3 mm. (C) Intraoperative image identifying the inguinal fat pad containing inguinal lymphatics (indicated by the open arrow), supplied by the superficial epigastric vessels. (D) Intraoperative image identifying the popliteal lymph node located within the inferior pole of the adductor thigh muscles (indicated by the closed arrow). (E) Intraoperative image of the implantable DDS placed in situ within the surgical wound. (F) Intraoperative image of the implantable DDS sutured to the fibers of the adductor thigh muscles.

Figure 3. Representative paw images after inducing hind limb lymphedema in treatment and placebo groups

Compared to control animals receiving placebo, the 9-cis RA group showed consistently less paw swelling as measured by electronic calipers. Subset of data used to plot Figure 4.

Figure 4. Sustained release 9-cis RA decreases paw swelling over time

Graphical representation of percent change in size of the operated paw relative to the unoperated paw. Compared to control animals, animals treated with 9-cis RA showed significantly reduced paw swelling at postoperative week 3 (7% mean difference, P=0.04), week 4 (12% mean difference, P=0.0002), week 5 (9% mean difference, P=0.0005), and week 6 (11% mean difference, P=0.0007). Within the treatment group, no significant difference in paw swelling was observed between time points.

Figure 5. Sustained release 9-cis RA results in faster lymphatic clearance

(A) Representative time-lapse images of ICG lymphography performed on postoperative day 42 (week 6). Subset of data used to plot Figure 5B. The accumulation of fluorescent signal seen at 1 hour post-ICG injection and persisting until hour 24 in the placebo group is consistent with dermal backflow, a characteristic finding of lymphedema on ICG lymphography due to disrupted lymphatic flow within dermal tissue. In 9-cis RA-treated animals, dermal backflow begins to disappear at hour 12, representing increased lymphatic clearance. (B) Change in fluorescence from injection time 0 plotted as a function of time (in arbitrary units). Compared to control animals, animals treated with 9-cis RA showed significantly faster lymphatic drainage as measured by decreased ICG fluorescence at 3, 6, 24, 48, 72, 96 and 144 hours following ICG injection (P<0.05).

Figure 6. Sustained release 9-cis RA decreases epidermal hyperplasia, collagen deposition, and lymphatic vessel size

(A) Representative picrosirius red stained cross-section of operated paw skin from a 9-cis RA-treated mouse. Horizontal scale bars represent 100 μm. (B) Representative picrosirius red stained cross-section of operated paw skin from a placebo-treated mouse. Note the decrease in collagen deposition in the operated paw skin of mice treated with 9-cis RA pellets. (C) Representative H&E stained cross-section of operated paw skin from a 9-cis RA-treated mouse. Horizontal scale bars represent 100 μm, vertical arrows represent Malpighian layer thickness. (D) Representative H&E stained cross-section of operated paw skin from a placebo-treated mouse. Note the decrease in hyperkeratosis and acanthosis in the operated paw skin of mice treated with 9-cis RA. (E) Representative LYVE-1 stained cross-section of operated paw skin from a 9-cis RA-treated mouse. Horizontal scale bars represent 100 μm, closed arrows indicate lymphatic vessels. (F) Representative LYVE-1 stained cross-section of operated paw skin from a placebo-treated mouse. Note the smaller lymphatic vessel diameter in the operated paw skin of mice treated with 9-cis RA. (G) Animals receiving 9-cis RA pellets showed a 15% decrease in collagen density per total tissue area compared to animals receiving placebo, indicating a reduced fibrotic response typical of lymphedema (P=0.10). (H) Animals receiving 9-cis RA pellets showed a 55% decrease in Malpighian layer thickness compared to animals receiving placebo (P=0.04), indicating reduced epidermal hyperplasia typical of lymphedema. (I) Animals receiving 9-cis RA pellets showed a 104% increase in number of LYVE-1+ lymphatic vessels compared to animals receiving placebo (P=0.04), indicating increased lymphatic density secondary to lymphangiogenesis. (J) Animals receiving 9-cis RA pellets showed a 52% decrease in LYVE-1+ lymphatic vessel diameter compared to animals receiving placebo (P=0.02), indicating preservation of the normal lymphatic architecture without luminal dilation.