Development of a mouse model for the visual and quantitative assessment of lymphatic trafficking and function by in vivo imaging

Abstract

Methods for quantitative analysis of long distance lymphatic transport of nanoparticles in live animals are yet to be established. We established a mouse model for analysis of time-dependent transport just beneath the abdominal skin to investigate lymph node-to-lymph node trafficking by in vivo imaging. For this purpose, popliteal lymph nodes (PLNs) as well as efferent and afferent lymphatic vessels, marginal veins, and feeding blood vessels were surgically resected to change the lymphatic flow from footpad injections. Using this model, we observed a novel lymphatic flow from the footpad to the proper axillary lymph node (ALN) via the inguinal lymph node (ILN). This drainage pathway was maintained over 12 weeks. Time-dependent transportation of 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide-labelled liposomes from the footpad to the ILN was successfully quantified by an in vivo imaging system. Moreover, congestion and development of a new collateral lymphatic route was visualised under a lymphedema status. Histological analysis of abdominal skin tissues of this model revealed that PLN resection had no effect on the abdominal lymphatic system between the ILN and ALN. These data indicate that this model might be useful to clarify the mechanisms of lymphedema and study direct transportation of lymph or other substances between lymph nodes.

Pre-collecting lymphatic vessels form detours following obstruction of lymphatic flow and function as collecting lymphatic vessels

“…In our study, detours were observed in the upper subcutaneous tissue after lymphatic ligation, and most detours appeared between two collecting LVs in the abdomen. These findings were consistent with those in our previous study [22]. Detours detected in the present study had Podoplanin + cells and valves, which were observed between capillary LVs in the dermis and collecting LVs in the lower subcutaneous tissue, and were connected to the collecting LVs.…”

“…The lymphatic detours we observed remained throughout the course of the study period. However, it was earlier than 15 days and later than 30 days before the lymphatic detours appeared after the ligation, while collateral LVs appeared from 1 to 4 weeks after LN dissection [13,15,19,21,22], indicating that our findings were consistent with those of previous studies.…”

“…In this study, one lymphatic detour was formed after ligations of the collecting LV at two points. However, Nakajima et al [21], Takeno and Fujimoto [15], Yamaji et al [22], Kwon and Proce [19], and Ikomi et al [13] showed that a network-like pattern of LVs and a few collateral LVs appeared after the LN dissection at least by week 1. Blume et al [14] reported that a collateral LV was sometimes observed after excision of only the popliteal LN; however, many collateral LVs appeared 1 week after the excision of popliteal LNs within the popliteal fat pad.…”

“…When collateral LVs bridging the distal LV (over the area of lymphadenectomy) to the proximal LV or to the remaining LNs with LVs are observed in patients using lymphoscintigraphy (at the area of the lymphadenectomy), lymphedema is said to be mild or moderate [3,5,7,[10][11][12]. Moreover, animal studies involving canines, rabbits, rats, and mice have previously reported that similar collateral LVs (also known as lymphatic detours) develop following LN dissection alone or in combination with LVs [13][14][15][16][17][18][19][20][21][22]. Collateral LVs in rodents have been detected using indocyanine green (ICG) in 2 days [22], in 3 days [20], in 1 week [14,15] and in 10 days [21] after lymphadenectomy.…”

“…Moreover, animal studies involving canines, rabbits, rats, and mice have previously reported that similar collateral LVs (also known as lymphatic detours) develop following LN dissection alone or in combination with LVs [13][14][15][16][17][18][19][20][21][22]. Collateral LVs in rodents have been detected using indocyanine green (ICG) in 2 days [22], in 3 days [20], in 1 week [14,15] and in 10 days [21] after lymphadenectomy. In our previous study, mice showed well-developed detours in the abdomen after lymphadenectomy of the inguinal LN [21].…”