Investigation of optimizing indocyanine green solution for in vivo lymphatic research using near-infrared fluorescence indocyanine green lymphangiography.

Despite the tireless efforts of many researchers in lymphatic research, indocyanine green (ICG) solution conditions suitable for lymphatic circulation tests have not been perfectly established yet. We aimed to investigate the optimal in vivo conditions of ICG solution to avoid photobleaching and quenching effects, which may affect the accuracy of lymphatic circulation evaluation. After ICG fluorescence intensity (or ICG intensity) was assessed under different in vitro conditions, the image quality of brachial lymph nodes (LNs) and collecting lymphatic vessels (LVs) in eight rats was investigated. The in vitro results showed that ICG intensity depends on concentration and time in various solvents; however, the brightest intensity was observed at a concentration of 8-30 µg/mL in all solvents. ICG concentration in the albumin (bovine serum albumin; BSA) solution and rat's plasma showed more than two times higher fluorescence intensity than in distilled water (DW) in the same range. However, saline reduced the intensity by almost half compared to DW. In the in vivo experiment, we obtained relatively high-quality images of the LNs and LVs using ICG in the BSA solution. Even at low concentrations, the result in the BSA solution was comparable to those obtained from high-concentration solutions commonly used in conventional circulation tests. This study provides valuable information about the conditions for optimal ICG intensity in near infrared fluorescence indocyanine green (NIRF-ICG) lymphangiography, which may be useful not only for the diagnosis of lymphatic circulation diseases such as lymphedema but also for preclinical research for the lymphatic system.

In Vivo Dynamic and Static Analysis of Lymphatic Dysfunction in Lymphedema Using Near-Infrared Fluorescence Indocyanine Green Lymphangiography.

BACKGROUND: Near-infrared fluorescence indocyanine green lymphangiography, a primary modality for detecting lymphedema, which is a disease due to lymphatic obstruction, enables real-time observations of lymphatics and reveals not only the spatial distribution of drainage (static analysis) but also information on the lymphatic contraction (dynamic analysis). METHODS: We have produced total lymphatic obstruction in the upper limbs of 18 Sprague-Dawley rats through the dissection of proximal (brachial and axillary) lymph nodes and 20-Gy radiation (dissection limbs). After the model formation for 1 week, 9 animal models were observed for 6 weeks using near-infrared fluorescence indocyanine green lymphangiography by injecting 6-µL ICG-BSA (indocyanine green-bovine serum albumin) solution of 20-µg/mL concentration. The drainage pattern and leakage of lymph fluid were evaluated and time-domain signals of lymphatic contraction were observed in the distal lymphatic vessels. The obtained signals were converted to frequency-domain spectrums using signal processing. RESULTS: The results of both static and dynamic analyses proved to be effective in accurately identifying the extent of lymphatic disruption in the dissection limbs. The static analysis showed abnormal drainage patterns and increased leakage of lymph fluid to the periphery of the vessels compared with the control (normal) limbs. Meanwhile, the waveforms were changed and the contractile signal frequency increased by 58% in the dynamic analysis. Specifically, our findings revealed that regular lymphatic contractions, observed at a frequency range of 0.08 to 0.13 Hz in the control limbs, were absent in the dissection limbs. The contractile regularity was not fully restored for the follow-up period, indicating a persistent lymphatic obstruction. CONCLUSIONS: The dynamic analysis could detect the abnormalities of lymphatic circulation by observing the characteristics of signals, and it provided additional evaluation indicators that cannot be provided by the static analysis. Our findings may be useful for the early detection of the circulation problem as a functional evaluation indicator of the lymphatic system.

Synthesis and in vitro characterizations of porous carboxymethyl cellulose-poly(ethylene oxide) hydrogel film.

BACKGROUND: Cellulose and its derivatives such as carboxymethyl cellulose (CMC) have been employed as a biomaterial for their diverse applications such as tissue engineering, drug delivery and other medical materials. Porosity of the scaffolds has advantages in their applications to tissue engineering such as more cell adhesion and migration leading to better tissue regeneration. After synthesis of CMC-poly(ethylene oxide) (PEO) hydrogel by mixing the solutions of both CMC-acrylate and PEO-hexa-thiols, fabrication and evaluation of a CMC-PEO gel and its film in porous form have been made for its possible applications to tissue regeneration. Physicochemical and biological properties of both CMC-PEO hydrogel and porous films have been evaluated by using physicochemical assays by SEM, FTIR and swelling behaviors as well as in vitro assays of MTT, Neutral red, BrdU, gel covering and tissue ingrowth into the pores of the CMC-PEO gel films. Degradation of CMC-PEO hydrogel was also evaluated by treating with esterase over time. RESULTS: Chemical grafting of acrylate to CMC was verified by analyses of both FTIR and NMR. CMC-PEO hydrogel was obtained by mixing two precursor polymer solutions of CMC-acrylate and PEO-hexa-thiols and by transforming into a porous CMC-PEO gel film by gas forming of ammonium bicarbonate particles. The fabricated hydrogel has swollen in buffer to more than 6 times and degraded by esterase. The results of in vitro assays of live and dead, MTT, BrdU, Neutral red and gel covering on the cells showed excellent cell compatibility of CMC-PEO hydrogel and porous gel films. Furthermore the porous films showed excellent in vitro adhesion and migration of cells into their pore channels as observed by H&E and MT stains. CONCLUSIONS: Both CMC-PEO hydrogel and porous gel films showed excellent biocompatibility and were expected to be a good candidate scaffold for tissue engineering.

Biocompatibility and resorption pattern of newly developed hyaluronic acid hydrogel reinforced three-layer poly (lactide-co-glycolide) membrane: histologic observation in rabbit calvarial defect model.

BACKGROUND: The aim of this study was to evaluate the biocompatibility and resorption pattern in three-layer poly (lactide-co-glycolide) (PLGA) membrane according to the concentrations of hyaluronic acid (HA) hydrogel in rabbit calvarial defect model. Four standardized circular defects with 8 mm diameter were created on the four rabbit calvarium. Three-layer PLGA membranes (5% and 10% HA gel) were used as the test groups, both collagen membrane and monolayer PLGA membrane as the control groups. RESULTS: After sacrificing the animals after 4 and 8 weeks, block sections were harvested and histological observation was performed. Pus formation was observed in a site on the three-layer PLGA membranes (with 10% HA gel) of 4 weeks group and initial inflammatory responses were observed on the three-layer PLGA membrane group. However, when compared to both the monolayer PLGA membrane group and collagen membrane group, the HA gel-reinforced three-layer PLGA membrane showed improved cell occlusion and retention period, showing the formation of the capsule-like structure. There was no definite difference between the results of the membranes fabricated with either 5% or 10% HA hydrogel. CONCLUSION: The HA reinforced three-layer PGLA membrane was retained longer than control group and showed good property in cell occlusion. Future study is under process to improve the inflammatory response of the three layer PLGA membranes, which were observed in this study.