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DISCUSSION
Part1
Lymphatic Fluid Stasis (LFS) and complete AND in a rat model (innovative posterior surgical approach)
A new model of Lymphatic Fluid Stasis (LFS) on the tail of a mouse resulting from impairment of lymphatic transport by traumatic tail surgery was proposed and used to study cells and mechanisms involved in subcutaneous tissue remodelling. However, AND was not performed in this model (Zampell et al., 2012). According to their initial results, LFS seemed to induce local oedema, local presence of various markers associated with fibrosis, adipogenesis and inflammation at 3 and 6 weeks post- intervention. To confirm these findings in a more physiologically relevant model, the authors performed AND in mice (N=6). Local oedema, Lymphatic Fluid Stasis and identical specific markers in the front paws of the mice were detected as well and only 3 weeks after surgery (Aschen et al., 2012).
Theoretically, in response to the highly traumatic surgery involving dissections of skin, connective tissue and muscles, an acute postoperative inflammatory process is produced and may last for several weeks. Such a persistent inflammatory process may partially have explained the above mentioned results. However, when AND in mice is compared to a sham surgical procedure, the results seem to indicate that only AND produces LFS and is responsible for the cited subcutaneous remodeling (Aschen et al., 2012).
To corroborate with these findings, an innovative posterior surgical approach was developed by the authors realising a minimal invasive and less traumatic procedure (Pastouret et al., 2016). According to the anatomy of a laboratory rat and for reaching the deep axillary nodes by a skin incision placed across the axilla, it was essential to cut the cutaneous trunci muscle as it is attached to the pectoralis muscle. Both protect the axilla and no direct access is present between both muscles to reach that site (Fig. 9).
Fig. 9 - Cutaneous trunci and pectoralis muscles in a rat following skin dissection.
Lateral views of a dissected front leg (arm and shoulder). The black arrow indicates the pectoralis muscle and the white arrow shows the cutaneous trunci muscle.
In another AND rat model, an anterior surgical approach was used to induce secondary lymphoedema by adding inflammatory drugs (Becker C, 1987; Mendez et al., 2012). The posterior surgical approach allowed to realise a complete AND without muscle dissection, no nervous lesion and avoiding infection (30/30).
The extended period of time following posterior surgery and before examination is an important factor (12 weeks) to evidence if postsurgical inflammation is avoided.
THE EUROPEAN JOURNAL OF LYMPHOLOGY - Vol. XXIX - Nr. 75 - 2017
An ICG mapping was selected to detect LFS and FLSP. By comparing the results in the study of Aschen (2012) to our presented findings, a difference regarding the evidence of LFS was shown. In the presented study, LFS never was detected at the operated sides 12 weeks following AND and no increase was observed in volume of the operated versus the non-operated distal front paw volume.
Skin sample collections were carried out to detect subcutaneous remodelling and sections were stained for subsequent analysis.
Regeneration process (lymphangiogenesis) and Functional Lymphatic Substitution Pathway (FLSP)
Lymphangiogenesis or the formation of new lymph vessels from pre-existing lymph vessels (Leclers et al., 2005) appears during processes of inflammation, metastasing tumours and wound healing (Tammela and Alitalo, 2010). Morphological and biochemical aspects of lymphangiogenesis during wound healing have been actively studied in the past and are topics of actual research (Lievens, 1978; Nogami et al., 2009; Pastouret et al., 2014). In the presented study, a regeneration process is the major way for lymphatic flow restoration following AND (73%), in combination with (30%) or without FLSP (43%). The FSLP found in our AND rat model is exclusively due to occurrence of lymphatic perforating vessels. During ICG mapping, functional anterior or posterior contralateral substitution lymphatic pathways were never observed. On the contrary, in another secondary lymphoedema rat model, contralateral substitution lymphatic pathways could well be observed during ICG mapping after lymphadenectomy (Takeno and Fujimoto, 2013).
In a preliminary study in a rat, lymphatic perforating vessels were observed for the first time in AND rats after Evans Blue dye injection (Fig. 10), skin and muscles dissection (Pastouret F. et al., 2016). Specific location was identified at the level of a precise point where the spinodeltoid muscles and the long and lateral portion of the triceps brachii meet (Pastouret et al., 2016). In the present study, perforating vessels were mapped in the same anatomic location during the second ICG mapping.
Perforating vessels were never observed on the non-operated sides in 50 mapped front paws in the control and experimental groups. The authors conclude that the described lymphatic perforating vessels following AND were not functional before surgery. In our rat model the experiments do not allow to explain the origin of these vessels which probably already existed since foetal period or were formed during lymphangiogenesis in the postsurgical phase. Our results regarding the volume change 12 weeks following surgery, seem to indicate that a regeneration process and FSLP play a primordial role to prevent LFS and secondary lymphoedema.
The change of paw volume is an important clinical sign. However, paw swelling is not sufficient to detect early secondary oedema. In the diagnosis, more attention is focussed on the presence of subclinical signs such as the significant changes in lymphatic patterns during the latent stage of lymphoedema, for example LFS, dermal back flow, without the clinical sign of swelling (Ogata et al., 2007).
In a secondary lymphoedema rat model, recovery of the lymphatic system following AND may lead to a chronic and latent lymphatic insufficiency without the clinical sign of swelling. The swelling associated with chronic lymphoedema may develop and appear
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