| Lymphatic malformation (LM) is a common benign mass in children and adults and may be representative of a derangement in lymphangiogenesis. These lesions have high recurrence rates and significant morbidity associated with surgery. Extensive microcystic malformations are difficult to eradicate.The pathogenesis of LM remains controversial, and there has been little progress in the understanding of this, which partly because of lacking effective research models for the LM. Evaluation of the efficacy of treatment strategies for LM requires a suitable animal model.Ideally, the model should replicate untreated human LM in its pathogenesis and pathological characteristics. Freund’s incomplete adjuvant (FIA) has been used to induce LM in mice and rats, however, the LM in these animal models, which were small and confined to the peritoneum, did not sufficiently simulate human oral and cervicofacial LM.In this study, we introduced LM in the neck and the floor of the mouth of the rats, this rat model simulated human oral and cervicofacial LM on the basis of the gross, microscopical and ultrastructural features of the lesions and on Raman spectral analysis of the cyst contents. We also compared the form and size of LM which were induced by different adjuvants, in order to investigate the possible mechanism of adjuvants. At the mean time, we explored the possible effects of vascular endothelial growth factor-C (VEGF-C) in the induction and development of LM.Part I Induction of Lymphatic Malformation in the neck and the floor of the mouth of Rats by Injection of Freund’s incomplete adjuvantObjective: To develop a highly reproducible method for the induction of lymphatic malformation in a rat model by injection of Freund’s incomplete adjuvant (FIA).Methods:16 female Wistar rats were randomly divided into four groups (group FIA-N, group FIA-F, group PBS and group BLANK). Group FIA-N received a subcutaneous injection of 0.2 ml FIA into the neck; group FIA-F received a submucosal injection of 0.2 ml FIA into the floor of the mouth; group PBS received both a subcutaneous injection of 0.2 ml PBS into the neck and a submucosal injection of 0.2 ml PBS into the floor of the mouth; and group BLANK with on treatment. Two weeks later, each group received the same dose of injections again. And then, the injection sites were monitored for the development at 2-week intervals for two months. After that, the rats were sacrificed and histological examination and transmission electron microscopy of the identified lesions were performed. Results:In group FIA-N, the rats developed translucent or transparent cystic lesions in the neck; in group FIA-F, the rats developed white translucent plaque-like lesions in the floor of the mouth; no cystic lesions or plaque-like lesions were detected in the rats of group PBS and group BLANK. By HE staining, the lesions in the neck and the floor of the mouth presented macrocysts and micricysts; immunohistochemical (IHC) examination revealed that the cysts were lined by endothelium, which expressed the lymphatic endothelial markers LYVE-1 and VEGFR-3. Transmission electron microscopy (TEM) revealed that the endothelia cells had no basement membrane or surrounding pericytes. Conclusion:The cystic lesions induced in the rats were consistent with human oral and cervicofacial LM. This rat model can sufficiently simulate human oral and cervicofacial LM.Part II The comparison of the LM in the neck of rats induced by different adjuvantsObjective:Comparing the form and size of the LM in the neck of rats which were induced by different adjuvants, in order to investigate the possible mechanism of the adjuvants in the induction and development of LM. Methods:28 female Wistar rats were randomly divided into seven groups (group FIA, group FCA, group MF59, group WO, group AH, group PBS and group BLANK). The former five groups received two subcutaneous injections of 0.2 ml different adjuvants (FIA, Freund’s complete adjuvant (FCA), MF59, White oil and Aluminium hydroxide) into the neck over a 2-week period, while group PBS received a subcutaneous injection of 0.2 ml PBS into the neck, and group BLANK with on treatment. After two months, the rats were sacrificed, measured the volume of the lesions in each group and histological examination was performed. At the mean time, the fluid content of macrocysts in group FIA and group FCA were aspirated for Raman Spectroscopy. Results:In group FIA, group FCA and group WO, the rats developed translucent or transparent macrocystic lesions in the neck; in group MF59, the rats developed few transparent microcystic lesions in the neck; in group AH, the injection sites were occurred of ulcers and scab, no cystic lesions were detected in the rats of group PBS and group BLANK. Comparing the volume of the lesions in each group, the lesions in group FIA and group FCA were significantly greater than the group WO and group MF59. By HE staining, the lesions in group FIA, group FCA and group WO presented macrocysts and micricysts; and in group MF59, only micricysts could be seen. IHC examination revealed that the cysts were lined by endothelium, which expressed LYVE-1 and VEGFR-3. The fluid contents aspirated from the cysts in group FIA and group FCA had similar stratification to FIA and FCA emulsions, and the Raman spectra of the cyst contents demonstrated the presence of protein and lipid, which were similar to the spectra of FIA and FCA emulsions in wave form and wave number.Conclusion:Different adjuvants (except for Aluminium hydroxide) can induce LM in rats. FIA and FCA can be used as first choice. The formation of macrocysts was due to the depot formation of adjuvants at the injection sites while microcysts may be induced by humoral and cellular immune responses.PartⅢThe role of vascular endothelial growth factor-C in the formation and growth of LMObjective: To investigate the role of vascular endothelial growth factor-C (VEGF-C) in the formation and the growth of LM. Methods:28 female Wistar rats were randomly divided into seven groups (group FIA, group FIA-V, group FCA, group FCA-V, group PBS-V, group PBS and group BLANK). The former five groups received two subcutaneous injections (FIA, FIA with VEGF-C, FCA, FCA with VEGF-C, PBS with VEGF-C) into the neck over a 2-week period, while group PBS received a subcutaneous injection of PBS into the neck, and group BLANK with on treatment. After two months, the rats were sacrificed, measured the volume of the lesions in each group and histological examination was performed. Results: In the former four groups, the rats developed translucent or transparent macrocystic lesions in the neck, while in group PBS-V, like group PBS and group BLANK, no cystic lesions were detected. More numerous and larger cystic lesions were induced in rats when VEGF-C was co-injected with adjuvants. By HE staining, the lesions of each group presented macrocysts and micricysts; IHC examination revealed that the cysts were lined by endothelium, which expressed LYVE-1 and VEGFR-3. Conclusion:The role of VEGF-C is to promote the formation and growth of LM rather than induce the formation of the LM. |
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