Friday, February 24, 2006

Postmastectomy Lymphedema: Long-term Results Following Microsurgical Lymph Node Transplantation

To Be Published: March 2006

Becker C, Assouad J, Riquet M, Hidden G.

>From Service de Chirurgie Thoracique, Hopital Europeen Georges Pompidou, Paris, France.


Lymphedema complicating breast cancer treatment remains a challenging problem. The purpose of this study was to analyze the long-term results following microsurgical lymph node (LN) transplantation.


Twenty-four female patients with lymphedema for more than 5 years underwent LN transplantation. They were treated by physiotherapy and resistant to it. LNs were harvested in the femoral region, transferred to the axillary region, and transplanted by microsurgical procedures. Long-term results were evaluated according to skin elasticity, decrease, or disappearance of lymphedema assessed by measurements, isotopic lymphangiography, and ability to stop physiotherapy.


The postoperative period was uneventful; skin infectious diseases disappeared in all patients. Upper limb perimeter returned to normal in 10 cases, decreased in 12 cases, and remained unchanged in 2 cases. Five of 16 (31%) isotopic lymphoscintigraphies demonstrated activity of the transplanted nodes. Physiotherapy was discontinued in 15 patients (62.5%). Ten patients were considered as cured, important improvement was noted in 12 patients, and only 2 patients were not improved.


LN transplantation is a safe procedure permitting good long-term results, disappearance, or a noteworthy improvement, in postmastectomy lymphedema, especially in the early stages of the disease.

PMID: 16495693 [PubMed - in process]


*** My concern with this procedure is: ***

Will taking lymph nodes from the femoral region and transplanting them elsewhere make the patient then susceptible to leg lymphedema.

Since this is a new experimental procedure, we have no long term followup.

Is this another "pig-in-a-poke" experiment putting the patients in even more danger?


Monday, February 20, 2006

Health Alert: Lymphedema study

(National) February 10, 2006 - One of the side effects of breast cancer treatment when lymph nodes are removed or scarred is lymphedema . It causes painful swelling in the arm near the affected, breast but there is a new study to help patients cope with the problem.

Valjean Waddy is a breast cancer survivor. She says, "When I've tried to buy clothes, one arm fits tighter. Some, some shirts that I have I can't wear."

Waddy hopes strength training will stop the swelling in her arm. The 43-year-old has lymphedema - fluid builds up in her left arm as a result of her cancer treatment. For years, women with the condition have been told to take it easy.

Dr. Kathryn Schmitz, "These women who have had breast cancer treatment are told not to lift anything heavier than five to fifteen pounds ever again in their lives." But Schmitz doesn't buy it, "If someone has had a damaged heart, do you tell them to sit down and not to do any exercise again? No."

So, women like Valjean are working out as part of a unique study at the University of Pennsylvania. Dr. Schmitz says, "What we're proposing is that twice-a-week strength training is actually safe for these women, healthful for them, health promoting."

Participants start out with the lightest weights and add on gradually. A pilot study shows the exercise is safe and participants reported an improvement in their symptoms.

Dr. Schmitz says, "They got a lot stronger, that their body fat percentage went down, their fitness improved and we found that their quality of life improved as well."
Lymphedema can be very painful and disfiguring. It can also interfere with wound healing and increase the risk of infection.

"It's a chronic condition," explains Dr. Schmitz. "It's not something that will ever go away once you are diagnosed. And the issue is keeping it at bay."

Depending on her findings, Dr. Schmitz hopes to organize an exercise program for lymphedema patients in the future.

Women interested in taking part in the study in Philadelphia can call 215-898-5112.

The National Lymphedema Network website is

Posted 4:52pm by Bryce Mursch


Saturday, February 18, 2006

T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema

EMBO J. 2003 July 15; 22(14): 3546–3556. doi: 10.1093/emboj/cdg342.

2003 European Molecular Biology Organization

Vivien Schacht, Maria I. Ramirez,1 Young-Kwon Hong, Satoshi Hirakawa, Dian Feng,2 Natasha Harvey,3 Mary Williams,1,4 Ann M. Dvorak,2 Harold F. Dvorak,2 Guillermo Oliver,3 and Michael Detmar5

Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, 1Pulmonary Center, Department of Medicine and 4Department of Anatomy, Boston University School of Medicine, Boston, MA 02118, 2Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215 and 3Department of Genetics, St Jude Children’s Hospital, Memphis, TN 38105, USA

5Corresponding author e-mail:

aV.Schacht and M.I.Ramirez contributed equally to this work

Received January 20, 2003; Revised May 13, 2003; Accepted May 19, 2003.


Within the vascular system, the mucin-type transmembrane glycoprotein T1α/podoplanin is predominantly expressed by lymphatic endothelium, and recent studies have shown that it is regulated by the lymphatic-specific homeobox gene Prox1. In this study, we examined the role of T1α/podoplanin in vascular development and the effects of gene disruption in mice. T1α/podoplanin is first expressed at around E11.0 in Prox1-positive lymphatic progenitor cells, with predominant localization in the luminal plasma membrane of lymphatic endothelial cells during later development. T1α/podoplanin–/– mice die at birth due to respiratory failure and have defects in lymphatic, but not blood vessel pattern formation. These defects are associated with diminished lymphatic transport, congenital lymphedema and dilation of lymphatic vessels. T1α/podoplanin is also expressed in the basal epidermis of newborn wild-type mice, but gene disruption did not alter epidermal differentiation. Studies in cultured endothelial cells indicate that T1α/podoplanin promotes cell adhesion, migration and tube formation, whereas small interfering RNA-mediated inhibition of T1α/podoplanin expression decreased lymphatic endothelial cell adhesion. These data identify T1α/podoplanin as a novel critical player that regulates different key aspects of lymphatic vasculature formation.

Keywords: angiogenesis/lymphangiogenesis/podoplanin/Prox1/T1α


The lymphatic vascular system maintains tissue fluid homeostasis and mediates the afferent immune response, but can also aid in the metastatic spread of malignant tumors (Detmar and Hirakawa, 2002). Dysfunction or abnormal development of cutaneous lymphatic vessels results in lymphedema, which is associated with defects in tissue repair and the immune response (Mallon and Ryan, 1994). Although the mechanisms that control the development of the blood vascular system have been well studied (Carmeliet, 2000), those of the lymphatic vessels are poorly understood.

Recent analyses of Prox1-deficient mice have shown that the lymphatic vascular system, as predicted by Sabin (1902), originates from the embryonic veins (Wigle and Oliver, 1999; Oliver and Detmar, 2002). Beginning at embryonic day (E) 9.5 of mouse development, the homeobox gene Prox1 is specifically expressed by a subpopulation of endothelial cells that are located on one side of the anterior cardinal vein. This is followed by polarized budding and migration of these Prox1-positive lymphatic progenitor cells, which eventually form lymphatic sacs and then the entire lymphatic vasculature. In Prox1-null mice, the budding and sprouting of lymphatic endothelial cells from the veins is arrested at ∼E11.5–E12.0, and these mice completely lack a lymphatic vascular system (Wigle and Oliver, 1999). We and others have shown recently that ectopic expression of Prox1 in primary human blood vessel endothelial cells represses the expression of several genes that are associated with the blood vascular phenotype (Hong et al., 2002; Petrova et al., 2002). Prox1 expression was also found to upregulate the expression of lymphatic-specific genes (Hong et al., 2002; Petrova et al., 2002), indicating its function as a master control gene that determines lymphatic endothelial cell fate (Oliver and Detmar, 2002). These studies identified the mucin-type transmembrane glycoprotein T1α/podoplanin as one of the primary Prox1-induced genes (Hong et al., 2002).

T1α/podoplanin is expressed by cultured human lymphatic endothelial cells, and is one of the most highly expressed lymphatic-specific genes (Petrova et al., 2002; Hirakawa et al., 2003). In vivo expression of T1α/podoplanin in lymphatic endothelium was first reported by Wetterwald et al. (1996), who named it ‘E11 antigen’. It was characterized further under the name ‘podoplanin’, because of its low level expression in kidney podocytes (Breiteneder-Geleff et al., 1997). Podoplanin is homologous to T1α, which was originally found to encode an antigen that is selectively expressed at the apical surface of alveolar type I cells in rat lung (Dobbs et al., 1988; Rishi et al., 1995). Expression of T1α has also been detected in the choroid plexus, ciliary epithelium of the eye, intestine, kidney, thyroid and esophagus of the fetal rat (Williams et al., 1996), and it has been shown to be homologous to the OTS-8 gene, a phorbol ester-induced gene in MC3T3-E1 mouse osteoblast cells (Nose et al., 1990). Other homologs include RTI40 (Gonzalez and Dobbs, 1998), murine gp38 (Farr et al., 1992), canine gp40 (Zimmer et al., 1997), human gp36 (Zimmer et al., 1999) and murine PA2.26 (Gandarillas et al., 1997).

There have been many studies of T1α/podoplanin expression in the lymphatic vascular system (Kriehuber et al., 2001; Maekinen et al., 2001; Hong et al., 2002; Petrova et al., 2002; Hirakawa et al., 2003). In spite of the large number of descriptive studies, little is understood about T1α/podoplanin’s biological function. We examined its role in lymphatic and blood vessel development by examining mice that have targeted deletions in the T1α/podoplanin gene (Ramirez et al., 2003).

Here, we show that within the vascular system, T1α/podoplanin is first expressed between E10.5 and E11.5 in endothelial cells of the cardinal vein and in budding, Prox1-positive lymphatic progenitor cells. T1α/podoplanin expression becomes specifically restricted to lymphatic endothelium during later development. Ultrastructural analysis revealed its predominant localization to the luminal plasma membrane of lymphatic vessels. We found that T1α/podoplanin–/– mice have defects in lymphatic vessel, but not blood vessel, pattern formation. These defects lead to diminished lymphatic transport, congenital lymphedema and dilation of cutaneous and intestinal lymphatic vessels. Overexpression of T1α/podoplanin in cultured vascular endothelial cells promoted the formation of elongated cell extensions and significantly increased endothelial cell adhesion, migration and tube formation. Together, these findings suggest that the transmembrane glycoprotein T1α/podoplanin is required to regulate key aspects of lymphatic vascular formation.


T1α/podoplanin is expressed by budding Prox1-positive lymphatic progenitor cells

In agreement with previous observations showing T1α/podoplanin expression in the central nervous system and the foregut at around E9 (Rishi et al., 1995; Williams, 2003), we detected expression of T1α/podoplanin in the neural tube of wild-type mice at E10.5 (Figure 1A). However, no vascular expression of T1α/podoplanin was detected yet at this time point, whereas the homeobox gene Prox1 was already expressed by a subset of endothelial cells of the anterior cardinal vein (Figure 1A and B). By E11.5 (data not shown) and E12.5, T1α/podoplanin was expressed by all endothelial cells of the cardinal vein and by the Prox1-positive lymphatic progenitor cells that had already budded off from the embryonic veins (Figure 1C and D). Two days later, T1α/podoplanin expression was restricted to the budded Prox1-positive lymphatic endothelial progenitor cells and to the Prox1-positive lymphatic endothelial cells that lined the developing primitive lymph sacs (Figure 1C and F). After birth, vascular T1α/podoplanin expression was almost exclusively detected in lymphatic vessels (see below).

Impaired lymphatic transport and formation of lymphedema in neonatal T1α/podoplanin-deficient mice

Mice with heterozygous and homozygous disruptions of the T1α/podoplanin gene (Ramirez et al., 2003) were compared with their wild-type littermates for all investigations. Whereas T1α/podoplanin+/– mice were healthy and fertile, and were macroscopically indistinguishable from their wild-type littermates, T1α/podoplanin–/– mice died immediately after birth, due to respiratory failure caused by impaired formation of alveolar airspace, associated with reduced numbers of differentiated type I alveolar epithelial cells in the lung (Ramirez et al., 2003). The skin of these mice was cyanotic and its texture was smoothened (Figure 2A). The lower limbs were markedly swollen, and thickened skin folds were clearly detectable in the neck area, indicative of cutaneous lymphedema (Figure 2A).

To investigate lymphatic transport, we intradermally injected Evans blue dye into the dorsum of the footpads of newborn mice. In both wild-type and T1α/podoplanin+/– mice, the dye was immediately transported through a dense network of interconnected dermal lymphatic capillaries and larger collecting lymphatic vessels towards the popliteal lymph nodes (Figure 2B and C). In T1α/podoplanin–/– mice, in contrast, only dilated lymphatic vessels were visible, and small dermal capillaries were not detectable (Figure 2D). Immediately after injection of Evans blue into all four extremities, retroperitoneal para-aortic lymph nodes and lymphatic ducts were clearly stained blue in wild-type and in T1α/podoplanin+/– mice, indicating efficient centripetal lymphatic transport (Figure 2E and F). Although immunofluorescence stainings revealed the presence of retroperitoneal lymphatic ducts in all of the investigated mice (data not shown), no staining of para-aortic lymphatic structures was detected in T1α/podoplanin–/– mice (Figure 2G), demonstrating that lymphatic transport was impaired.

Dilation of intestinal and cutaneous lymphatic vessels, but not of blood vessels, in T1α/podoplanin-null mice

Both the skin and intestine are characterized by their rich lymphatic vascularization, and these tissues are highly sensitive to impairment of lymphatic network formation. Using differential immunostains for the lymphatic-specific hyaluronan receptor LYVE-1 (Prevo et al., 2001) and the endothelial plasma membrane molecule CD31, we found several slightly enlarged submucosal lymphatic vessels in the small intestine of T1α/podoplanin+/– mice as compared with wild-type mice (Figure 3A and E). In T1α/podoplanin–/– mice, an increased number of severely dilated submucosal lymphatic vessels was found (Figure 3I), whereas no major alterations of subserosal lymphatic capillaries were detected. LYVE-1-positive lacteals were completely absent in T1α/podoplanin–/– mice (Figure 3I), whereas these formed normally in wild-type and T1α/podoplanin+/– mice (Figure 3A and E, asterisks). The number and size of CD31-positive/LYVE-1-negative intestinal blood vessels, in contrast, were comparable in all genotypes (Figure 3A, B, E, F, I and J).

The T1α/podoplanin–/– mice also had enlarged lymphatics in the skin, as compared with wild-type and T1α/podoplanin+/– mice (Figure 3C, G and K). Computer-assisted morphometric image analyses confirmed that the average area of dermal LYVE-1-positive lymphatic vessels was significantly increased in T1α/podoplanin–/– mice (Figure 3N), whereas no differences in the size of blood vessels were found (Figure 3M). Immunofluorescence analysis of the intestine and the skin confirmed that lymphatic vessels in both wild-type and T1α/podoplanin+/– mice strongly expressed T1α/podoplanin. In contrast, no immunoreactivity was detected in T1α/podoplanin–/– mice, confirming the complete disruption of this gene and the specificity of the 8.1.1 antibody, that was originally raised against the gp38 antigen (Farr et al., 1992), for T1α/podoplanin (Figure 3B, D, F, H, J and L).

T1α/podoplanin deficiency results in impaired patterning of lymphatic capillary networks

We investigated whether T1α/podoplanin deficiency, in addition to causing the enlargement of intestinal and cutaneous lymphatic vessels, might also affect the patterning of the lymphatic network in the same anatomical regions. To this end, we performed immunostain analysis of LYVE-1 expression on tissue samples from the intestine (ileum) and from the ear skin of all newborn genotypes. We observed a dense and well-organized network of intestinal lymphatic capillaries in wild-type and T1α/podoplanin+/– newborn mice (Figure 4A and E).

T1α/podoplanin–/– newborn mice, in contrast, developed areas of extremely enlarged lymphatic vessels, and the pattern of these vessels was completely disorganized (Figure 4I). Analysis of the ear skin of wild-type and T1α/podoplanin+/– mice revealed well-organized networks of LYVE-1-positive lymphatic capillaries (Figure 4B and F). Most of these capillaries were interconnected, and only a few blind beginning lymphatic capillaries were detected (Figure 4B and F). In contrast, the formation of lymphatic capillary networks was impaired in the ear skin of T1α/podoplanin–/– mice. These mice possessed an increased number of non-anastomozing, blind beginning cutaneous lymphatic capillaries (Figure 4J, arrowheads), indicative of impaired lymphatic network patterning.

Because there were no clear-cut differences in lymphatic network patterns between newborn T1α/podoplanin+/– and wild-type mice, we performed immunohistochemical analyses on intestine (ileum) and ear skin tissue obtained from adult (4-month-old) T1α/podoplanin+/– mice and their littermates. Although the defects in lymphatic network patterning were not as striking as those seen in newborn T1α/podoplanin–/– mice, there were areas of dilated lymphatic vessels in the ear skin (Figure 4H) and in the intestine of T1α/podoplanin+/– mice, in addition to incomplete network formation (Figure 4G) that was not observed in wild-type littermates (Figure 4C and D).

Ultrastructural localization of T1α/podoplanin in intestinal lymphatic vessels, but not in blood vessels

To investigate the ultrastructural localization of T1α/podoplanin in the vascular system, we performed immuno-nanogold electron microscopy, using intestinal tissue samples obtained from newborn mice. In wild-type mice, high levels of membrane-bound T1α/podoplanin were detected at the luminal side of intestinal lymphatic vessels (Figure 5A). Fewer nanogold particles were observed on the abluminal plasma membrane, and no labeling was detected within the cytoplasm (Figure 5A). Occasionally, the lateral plasma membranes between adjacent cells were also labeled. T1α/podoplanin expression was completely absent from blood vessel endothelial cells in the intestine of newborn wild-type mice (Figure 5B). Lymphatic endothelial cells in the small intestine of T1α/podoplanin–/– mice were not immunolabeled by the anti-T1α/podoplanin antibody (Figure 5D), confirming the disruption of the T1α/podoplanin gene in these mice.

T1α/podoplanin deficiency does not alter the ultrastructural architecture of intestinal lymphatic vessels or the distribution of LYVE-1 expression

We next investigated whether loss of T1α/podoplanin might result in abnormal ultrastructural architecture of lymphatic vessels or in altered distribution of the LYVE-1. We detected high levels of immuno-nanogold labeling for LYVE-1 at both the luminal and the abluminal plasma membrane of lymphatic endothelium in the intestine of newborn wild-type mice (Figure 6A). Some labeling of the lateral plasma membranes was also observed, with the exception of contact areas between adjacent cells. LYVE-1 expression was completely absent from blood vessel endothelium (Figure 6B), confirming the specificity of LYVE-1 for lymphatic endothelium. Lymphatic endothelial cells of T1α/podoplanin–/– mice also showed strong LYVE-1 immunogold labeling at both the luminal and the abluminal plasma membranes at levels similar to those observed in wild-type mice (Figure 6C and D). No ultrastructural differences in lymphatic vessel structure were observed between the different genotypes.

T1α/podoplanin deficiency does not impair epidermal differentiation

We occasionally detected T1α/podoplanin expression in basal keratinocytes of the epidermis in wild-type and T1α/podoplanin+/– mice (Figure 3D and H). To determine whether T1α/podoplanin deficiency might also affect epidermal structure or differentiation, we studied the expression of several markers of epidermal differentiation. A comparable expression pattern of keratin 14 (K14), which is expressed by proliferative basal keratinocytes (Fuchs and Byrne, 1994), was found in all three groups (Figure 3O, R and U). Expression patterns of the early and late epidermal terminal differentiation markers K10 (Figure 3P, S and V) and loricrin (Figure 3Q, T and W) were also similar in the skin in all three genotypes. Computer-assisted morphometric image analyses demonstrated comparable thickness of the epidermis in all genotypes (data not shown), and no major histological differences of epidermal structure were detected, indicating that T1α/podoplanin deficiency does not affect the formation or structure of the epidermis.

T1α/podoplanin promotes endothelial cell migration, adhesion and tube formation

To characterize further the biological function of T1α/podoplanin, we isolated lymphatic endothelial cells from the skin of newborn mice of all three genotypes. However, we were unable to expand lymphatic endothelial cell cultures obtained from all genotypes, even after addition of recombinant vascular endothelial growth factor (VEGF)-C to the growth medium (data not shown). We therefore decided to test the effects of T1α/podoplanin overexpression. Immortalized human microvascular endothelial cells (HMEC-1) were stably transfected with a pIRES2-rat T1α/podoplanin expression vector and pooled cells were used for subsequent in vitro studies. Immunostains revealed high levels of rat T1α/podoplanin protein in the stably transfected cells (Figure 7A) that projected extremely long and thin cell extensions, which were not seen in control cells that did not express rat T1α/podoplanin (Figure 7B and C). Some of the stably T1α/podoplanin-transfected cells also formed phalloidin-positive F-actin bundles at the periphery (Figure 7B and C), indicating that T1α/podoplanin expression might control endothelial cell cytoskeletal organization. We next studied whether overexpression of T1α/podoplanin could affect endothelial cell migration or adhesion. We found a >2-fold (P <>7D). T1α/podoplanin-overexpressing HMEC-1 cells also adhered more tightly to immobilized fibronectin (P <>7E).

To confirm these results further in a second, independent cell line, murine hemangioendothelioma-derived EOMA cells were stably transfected with the same pIRES2-rat T1α/podoplanin–enhanced green fluorescent protein (EGFP) construct or with the control vector. Three clones with high expression of the transfected T1α/podoplanin, along with three control clones, were used for subsequent experiments. Overexpression of T1α/podoplanin significantly enhanced EOMA cell migration towards type I collagen and also promoted cell adhesion in all three clones tested, as compared with the control clones (Figure 8A and B). T1α/podoplanin-overexpressing EOMA cell clones also showed a significantly increased ability to form tube-like structures after plating onto Matrigel (Figure 8C–E).

Inhibition of T1α/podoplanin expression by siRNA transfection reduces cell adhesion of human lymphatic endothelial cells

To investigate further the role of T1α/podoplanin in cell adhesion, we studied the effects of small interfering RNA (siRNA)-mediated inhibition of endogenous T1α/podoplanin expression in cultured human dermal lymphatic endothelial cells that are characterized by high expression levels of T1α/podoplanin (Hirakawa et al., 2003). Two out of three siRNAs tested efficiently reduced endogenous T1α/podoplanin protein expression by 49 and 34%, respectively, at 4 days after transfection, whereas the expression of another lymphatic marker, LYVE-1, remained unchanged (Figure 9A). Accordingly, lymphatic endothelial cell adhesion to type I collagen, which is closely associated with lymphatic vessels in vivo (Skobe and Detmar, 2000), was significantly inhibited by both of these siRNAs, as compared with control or sham-transfected control cells, at 4 days after transfection (Figure 9B).


Previous studies have identified T1α/podoplanin as a Prox1-induced gene (Hong et al., 2002) that is predominantly expressed, within the vascular system, in lymphatic endothelium (Wetterwald et al., 1996; Kriehuber et al., 2001; Maekinen et al., 2001; Hirakawa et al., 2003). We show that similarly to VEGF receptor-3 (Wigle et al., 2002), another Prox1 target gene (Hong et al., 2002; Petrova et al., 2002), T1α/podoplanin is expressed throughout the endothelium of the anterior cardinal vein at E12.5 of embryonic mouse development and later becomes restricted to the budding Prox1-positive lymphatic progenitor endothelial cells and to lymphatic endothelial cells of the embryonic lymph sacs and of lymphatic vessels. In contrast to Prox1-null mice, which fail to develop any lymphatic vasculature (Wigle and Oliver, 1999), T1α/podoplanin–/– mice develop a peripheral lymphatic vascular system; however, it exhibits pronounced defects in lymphatic vascular organization and function. Our findings indicate that T1α/podoplanin is important to regulate different aspects related to the later stages of lymphatic development and patterning. In contrast to other recently described gene targeting models, including mice deficient for angiopoietin-2 (Gale et al., 2002) or VEGF receptor-3 (Dumont et al., 1998), T1α/podoplanin deficiency selectively affects the lymphatic vascular system without any detectable effect on the development of the blood vascular system. Similarly to neuropilin-2 mutant mice which show only reduction of small lymphatic vessels but no alteration of the blood vascular system (Yuan et al., 2002), our findings in T1α/podoplanin-deficient mice are in agreement with the relatively late onset of T1α/podoplanin expression during vascular development, and with its predominant expression on lymphatic vascular endothelium.

Importantly, T1α/podoplanin–/– mice were characterized by congenital lymphedema, as manifested by the pronounced swelling of the limbs at birth. Intradermal dye injection into the foot pads of T1α/podoplanin–/– mice revealed several enlarged, plump lymphatic vessels, but failed to visualize the characteristic dermal capillary networks seen in wild-type and in T1α/podoplanin+/– mice. These networks were most probably filled from deeper lymphatic vessels through anastomozing vessels that still lack valves at the early stages of pre- and post-natal lymphatic development (Polonskaja, 1935). Because histological examination revealed the presence of dermal capillaries in T1α/podoplanin–/– mice, these findings indicate an insufficient formation of anastomozing lymphatic vessels between the superficial and subcutaneous lymphatic networks.

In the intestine of T1α/podoplanin–/– mice, lacteals were not detectable and many of the lymphatics of the submucosal plexus were enlarged. The physiological consequences of these developmental defects on the uptake of lipids from the intestine are not known, since these mice died immediately after birth (before the first feeding) and chyle transport could not be investigated. However, as previously described (Gale et al., 2002), angiopoietin-2-null mice develop chylous ascites shortly after birth, due to insufficient formation of lacteals. Because the observed morphological defect in T1α/podoplanin–/– mice is even more pronounced, we expect that lipid uptake in the intestine would also be defective. The observed enlargement of both dermal and submucosal intestinal lymphatics of T1α/podoplanin–/– mice is most likely to be related to the lack of connecting lymphatics between the superficial and deep networks, indicating that T1α/podoplanin is required for the formation of these specific lymphatic vessels.
We were unable to propagate lymphatic endothelial cells that were isolated from both wild-type and T1α/podoplanin–/– neonatal mice by using a modification of our recently established purification method for human dermal lymphatic endothelial cells (
Hirakawa et al., 2003). At present, there are no published reports on the successful propagation of primary murine lymphatic endothelial cells, and suitable culture techniques still remain to be established. Therefore, we investigated the cellular effects of T1α/podoplanin overexpression in two types of immortalized human and murine vascular endothelial cells that express only moderate levels of T1α/podoplanin (Hong et al., 2002). The induction of elongated endothelial cell extensions and cytoskeletal reorganization, together with the enhanced adhesion and migration of stably T1α/podoplanin-transfected endothelial cells are in agreement with its effects on the motility of immortalized keratinocytes (Scholl et al., 1999) and indicate that this protein controls endothelial cell functions that are required for normal lymphatic patterning during development. These findings were confirmed further by the reduced endothelial cell adhesion observed after inhibition of endogenous T1α/podoplanin expression by siRNA transfection. Because efficient cell migration is dependent upon the polarity of the migrating cells, and since T1α/podoplanin is also expressed on the apical surface of polarized alveolar epithelial cells (Dobbs et al., 1988; Rishi et al., 1995), it may exert an important role for the polarization of cells and for the stabilization of cellular protrusions at the leading edge of migrating cells. This proposed function is supported further by our findings that overexpression of T1α/podoplanin in endothelial cells promoted the formation of tube-like structures on Matrigel which provide a link to the in vivo findings of incomplete lymphatic network formation in T1α/podoplanin–/– mice.
T1α/podoplanin is a mucin-type glycoprotein with extensive O-glycosylation and a high content of sialic acid (
Gonzalez and Dobbs, 1998). This negatively charged structure probably is resistant to proteases and provides a physical barrier that protects cells from environmental agents (Zimmer et al., 1999). Immuno-nanogold electron microscopy showed that T1α/podoplanin is predominantly localized to the apical, luminal plasma membrane of intestinal lymphatic endothelial cells. This localization is similar to that reported for other cell types (Dobbs et al., 1988; Rishi et al., 1995; Williams et al., 1996; Breiteneder-Geleff et al., 1997; Zimmer et al., 1997) and is compatible with a protective function towards the proteinase-containing lymph (Bartos et al., 1979). It is of interest that T1α/podoplanin is also expressed by alveolar epithelial cells, cells of the choroid plexus, ependymal epithelia and mesothelia that are also exposed to an external or internal fluid compartment.

In conclusion, we propose that T1α/podoplanin is required to control different aspects of normal lymphatic vasculature formation. Lack of T1α/podoplanin leads to alterations in the final patterning of the lymphatic vasculature as well as in lymph transport. The future generation of a conditional inactivation of T1α/podoplanin will be a valuable tool to understand further its role in the normal function and in pathological alterations of the lymphatic vasculature.

Material and Methods

Generation of T1α/podoplanin knockout mice

T1α/podoplanin mutant mice were generated as described (Ramirez et al., 2003). Fragments were designed to replace ∼1.5 kb of the promoter sequence, 210 bp of the untranslated region (UTR), the first exon (67 bp) and 181 bp of the first intron. The efficiency of the disruption of the T1α/podoplanin gene was confirmed by northern and western blot analyses of newborn lung RNA and protein extracts that demonstrated the absence of T1α/podoplanin mRNA or protein in T1α/podoplanin–/– mice and reduced T1α/podoplanin mRNA levels in T1α/podoplanin+/– mice (Ramirez et al., 2003).

Tissue processing and immunostaining

Tissue samples were obtained from newborn T1α/podoplanin (+/+), (+/–) and (–/–) littermates that had been killed by intraperitoneal injection of barbital sodium within 10 min after birth and from wild-type embryos at days E10.5, E12.5 and E14.5, and were fixed in 4% paraformaldehyde (Fluka, Buchs, Switzerland). Immunofluorescence stains were performed on 6 µm cryostat sections of dorsal skin and of ileum, as well as on 10 µm sections of wild-type mouse embryos as described (
Wigle et al., 2002), using polyclonal rabbit antibodies against Prox1 (Wigle et al., 2002) and LYVE-1 (Prevo et al., 2001; kindly provided by Dr D.Jackson, Oxford, UK), a monoclonal rat antibody against CD31 (BD Pharmingen, San Diego, CA), a hamster antibody against murine gp38 (Farr et al., 1992; clone 8.1.1, Developmental Studies Hybridoma Bank, University of Iowa), antibodies against murine K14, K10 and loricrin (Babco, Richmond, CA) and corresponding secondary antibodies labeled with AlexaFluor488 or AlexaFluor594 (Molecular Probes, Eugene, OR). Cell nuclei were counterstained with 20 µg/ml Hoechst bisbenzimide (Molecular Probes). Whole-mount samples of mouse ears and of ileum were stained as described elsewhere (Gale et al., 2002). The tissues were examined by using a Nikon E-600 microscope (Nikon, Melville, NY) and images were captured with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Computer-assisted morphometric analysis of lymphatics and blood vessels within the dermis of eight animals per group (three fields per sample at 20× magnification) were performed as described previously (Streit et al., 1999), using the IP-LAB software (Scanalytics, Fairfax, VA). The differences in vessel size were analyzed by the two-sided unpaired Student’s t-test.

Immuno-nanogold electron microscopy

Small intestine samples were collected from genetically modified mice and their wild-type littermates immediately after birth. Tissue samples were fixed in 4% paraformaldehyde and transferred to 30% sucrose/phosphate-buffered saline (PBS). After embedding the tissues in OCT compound (Miles, Elkhart, IN), cryostat sections (5 µm) were immersed in 50 mM glycine. After washing, sections were immersed in normal goat serum (Vector Laboratories, Burlingame, CA) and incubated with anti-LYVE-1 or 8.1.1 antibody, followed by incubation with goat anti-rabbit Fab′, conjugated with 1.4 nm nanogold particles (Nanoprobes, Stony Brook, NY), or with goat anti-hamster IgG, conjugated with 0.8 nm nanogold particles (Aurion, Wageningen, The Netherlands). Sections were post-fixed in 1% PBS-buffered glutaraldehyde, developed with HQ silver enhancement solution (Nanoprobes), fixed in 5% sodium thiosulfate, post-fixed in 1% osmium tetroxide in sym-collidine buffer, washed in 0.05 M sodium maleate buffer, and stained with 2% uranyl acetate in 0.05 M sodium maleate buffer. Tissues were dehydrated in graded ethanols, infiltrated with propylene oxide–eponate (Ted Pella, Redding, CA) and embedded by inverting eponate-filled plastic capsules over the slide-attached tissue sections. After polymerization, eponate blocks were separated from the glass slides and thin sections were cut on an ultratome (Reichert Ultracuts, Austria), collected on uncoated 200 mesh copper grids (Ted Pella) and examined with a CM 10 transmission electron microscope (Philips, Eindhoven, The Netherlands). Specificity controls included replacement of the primary antibody with irrelevant rabbit IgG and omission of the specific primary antibody.

Cell culture and transfection

Immortalized HMEC-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) that contained 10% fetal bovine serum (FBS), 2 mM l-glutamine and antibiotics (Life Science, Grand Island, NY). The murine hemangioendothelioma-derived cell line EOMA (
Obeso et al., 1990) was maintained in DMEM containing 20% FBS and 4 mM l-glutamine. Primary human dermal lymphatic endothelial cells were isolated and propagated as recently described (Hirakawa et al., 2003). The rat T1α/podoplanin coding sequence (Rishi et al., 1995; GenBank accession No. U07797) was cloned into a pIRES2-EGFP vector (Clontech, Palo Alto, CA) which contains a cytomegalovirus (CMV) enhancer promoter and a neomycin selection cassette. The nucleotide sequence was verified using the Applied Biosystems Big Dye Terminator kits. After linerarization with BsaI, HMEC-1 and EOMA cells were transfected either with the full-length rat T1α/podoplanin cDNA or with pIRES2-EGFP vector alone using the SuperFect transfection reagent (Qiagen, Chatsworth, CA). Stably transfected cells were selected in growth medium containing 200 µg/ml neomycin (Gibco, Carlsbad, CA). For all experiments, pooled transfected HMEC-1 cells and three clones of stably transfected EOMA cells were used for T1α/podoplanin overexpression and for vector controls. To determine T1α/podoplanin expression levels, we performed SYBR Green-based real-time RT–PCRs as described (Hong et al., 2002), using the ABI Prism 7000 Sequence Detection System. The following forward and reverse primers were used: 5′-GACATGGTGAACCCAGGTCT-3 and 5′-AATGGGAGGCTGTGTTGGTA-3. Total RNAs were isolated using Tri-reagent (Sigma, St Louis, MO) and were treated with RNase-free RQ-DNase (Promega, Madison, WI). A 100 ng aliquot of RNA was used for each reaction. SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used for reactions with the addition of MultiScribe reverse transcriptase (Applied Biosystems). Protein expression was confirmed by immunofluorescence staining with a specific monoclonal anti-rat T1α/podoplanin antibody (Wetterwald et al., 1996). For F-actin staining, phalloidin–tetramethylrhodamine B isothiocyanate conjugate (Sigma) was used (Scholl et al., 1999).

Cell transfection with human T1α/podoplanin siRNAs

The following siRNA oligonucleotides were synthesized by Dharmacon (Lafayette, CO): (R1) 5′-GCGAAGACCGCUAUAAGUCdTdT-3′, (R2) 5′-AAGAUGGUUUGUCAACAGUdTdT-3′ and (R3) 5′-AGAUGA CACUGAGACUACAdTdT-3′. Primary human lymphatic endothelial cells (
Hirakawa et al., 2003) were transfected or not with siRNA oligonucleotides (500 nmol) or with equimolar concentrations of control plasmid vector by using the Nucleofector kit (Amaxa, Cologne, Germany) according to the manufacturer’s instructions. Cells were harvested at 2 and 4 days after transfection. For western analyses, cell lysates were obtained as described (Hong et al., 2002) and 30 ng of protein per sample were immunoblotted with an antibody against human T1α/podoplanin (Zimmer et al., 1997; kindly provided by Dr G.Herrler, Hanover, Germany) and with an antibody against human LYVE-1 (kindly provided by Dr D.Jackson, Oxford, UK).

Cell migration, adhesion and tube formation assays

For cell migration assays, 24-well FluoroBlok inserts (Falcon, Franklin Lakes, NJ; 8 µm pore size) were coated on the underside with 10 µg/ml fibronectin (BD Bioscience, Bedford, MA) or with 50 µg/ml type I collagen (Vitrogen, Palo Alto, CA) for 1 h, followed by addition of 100 µg/ml bovine serum albumin (BSA; Sigma) to block the remaining protein-binding sites. Cells (2 × 105 cells/ml) were seeded in serum-free EBM-2 medium (Clonetics, Watersville, MD) containing 0.2% de-lipidized BSA into each well and cells were incubated for 3 h at 37°C. Cells on the underside of inserts were stained with Calcein AM (Molecular Probes), and the fluorescence intensity was measured using the Victor2 Fluorometer (PerkinElmer, Boston, MA). Cell adhesion assays were performed as described (
Streit et al., 1999). Twelve-well plates were coated with 10 µg/ml fibronectin or with 50 µg/ml type I collagen for 1 h at 4°C, followed by blocking with 100 µg/ml BSA. Cells (1 × 105 cells in 200 µl of serum-free DMEM) were added to each well and were incubated at 37°C for 20 min. Unattached cells were removed by three gentle washes with PBS; attached cells were fixed with 4% paraformaldehyde, stained with Hoechst bisbenzimide and the number of attached cells/mm2 was determined. Tube formation assays were performed on Matrigel-coated 24-well plates (BD Bioscience) as described previously (Obeso et al., 1990). EOMA cells were seeded at a density of 2 × 105 cells/ml in growth medium and were incubated for 24 h at 37°C. After fixation with 4% paraformaldehyde, images were captured and the total length of tube-like structures per area was measured using the IP-LAB software. All studies were performed in triplicate. Statistical analyses were performed using the unpaired Student’s t-test.

Article with Acknowledgements

Monday, February 13, 2006

Lymphedema Therapy in the Vascular Anomaly Patient

Dec 2005, Vol. 3, No. 4: 253-255
Stanley G. Rockson, M.D.

Stanford Center for Lymphatic and Venous Disorders, Division of Cardiovascular Medicine, Stanford University School of Medicine, Falk Cardiovascular Research Center, Stanford, California.

Lymphedema Therapy in the Vascular Anomaly Patient:
Therapeutics for the Forgotten Circulation


Any discussion of lymphatic dysfunction and therapeutics must concern itself initially with the role of the lymphatic circulation in health and disease.1 In normal physiology this system is concerned with the flux of lymph from the extracellular space which, in turn, contributes to the maintenance of total body homeostasis.

A second, vital function is tied to the transport of leukocyte and antigen-presenting cells to lymphoid organs.2 Finally, the gastrointestinal lymphatics play an integral role in the nutritive absorption of lipids from the intestinal lumen.

In disease, the lymphatic system can be afflicted by either congenital (heritable) or acquired pathology. In most instances, including those that prevail in the vascular anomalies, disease is accompanied by the presence of either regional or generalized lymphedema, a condition that supervenes when there in an inherent imbalance between the formation of interstitial fluid and its absorption, as lymph, into the lymphatic vessels.

In the presence or absence of associated vascular anomalies, structural abnormalities in the lymphatic conduits will have predictable effects upon functional derangements and disease manifestations.3 Lymphatic aplasia, hypoplasia, and primary valvular insufficiency produce lymphatic hypertension, decreased contractility, and secondary valvular insufficiency. Obliteration or disruption of lymph vessels, as might occur in proliferative lesions of this vasculature, will lead to lymph stasis, with accumulation of lymph, retained
interstitial proteins, and glycosaminoglycans in the skin and subcutaneous structures.

With chronicity, these changes are accompanied by disruption of elastic fibers and activation of keratinocytes, fibroblasts, and adipocytes. The resultant stimulation of collagen production leads to thickening of the skin, subcutaneous tissue overgrowth, and fibrosis.

In addition to the chronic edema and fibrosis of lymphedema, lymph stasis has numerous additional longterm attributes: lymphostasis in the arterial wall, in concert with other vascular responses, leads to local arteriovenous flow disturbances; similar effects are seen in the ligaments and tendons; and disturbed immune traffic, in concert with the physical derangements in the tissues, predisposes to frequent, recurrent episodes of bacterial
soft tissue infection (cellulitis).

A consideration of lymphatic dysfunction in the context of vascular anomalies raises the question of the heritable nature of certain lymphatic disorders. In addition to the variety of chromosomal aneuploidy that has been described (trisomy 13, 18, 21, triploidy, Klinefelter syndrome, Turner syndrome), various dysmorphogenic genetic abnormalities have been associated with lymphedema and complex vascular anomalies: Noonan Syndrome; Noone-Milroy Hereditary Lymphedema; Meige Lymphedema (Lymphedema Tarda); Lymphedema Distichiasis; Cholestasis-lymphedema syndrome (Aagenaes syndrome);4 Lymphedema- microcephaly-chorioretinopathy;5 Neurofibromatosis type I (von Recklinghausen); Lymphedema-Hypoparathyroidism Syndrome; and Klippel-Trenaunay-Weber Syndrome.2,6

Lymphedema, even when superimposed upon a more complex vascular presentation, is most often readily identified by its physical characteristics, including pitting edema, peau d’orange, cutaneous fibrosis, and positive ‘Stemmer’ sign;7,8 where doubt exists, the presence of lymphatic vascular insufficiency can be verified by indirect radionuclide lymphoscintigraphy.3,9

Once identified, the lymphatic component of a vascular anomaly presentation mandates an aggressive physiotherapeutic approach that will require life-long emphasis. It has been proposed that meticulous measures designed to reduce edema may reduce the likelihood of disease progression and reduce the frequency of soft-tissue infection.10

Decongestive lymphatic therapy is an empirically derived, effective, multi-component technique for the reduction of volume of the involved limb and for the maintenance of integumentary health. It has been suggested that this method accelerates the transport of lymph and, thereby, facilitates the dispersal of retained interstitial protein.11

Decongestive lymphatic therapy integrates meticulous skin care, exercise, and the use of compressive elastic garments with daily application of a specific massage technique known as manual lymphatic drainage.10

The manual technique emphasizes the recruitment of watershed pathways for lymphatic flow through stimulation of edema-free zones of the trunk and uninvolved extremities; the mild tissue compression that is accomplished during manual lymphatic drainage enhances filling of the initial lymphatics and augments transport capacity through the cutaneous lymphatic dilatation and the development of accessory lymph collectors.12

Manual lymphatic drainage (MLD) should not be confused with other forms of therapeutic massage that have no effect on lymphatic contractility. Adjunctive to external compressive techniques in the limb, MLD will represent the mainstay of therapy for involvement of the head, neck, breast, and genitalia, where the external application of ancillary compressive devices and garments is difficult or impossible.

Where feasible, however, it is essential to maximize compression for edema volume to be effectively reduced. In the acute treatment phase of limb lymphedema, this compression therapy will take the form of multi-layer short stretch bandage application, which has been demonstrated to augment the effect of joint/muscle pump upon lymph transit through the limb;13 furthermore, as tissue pressure is increased, there is a reduction in the abnormally increased ultrafiltration which, in turn, leads to improved fluid reabsorption.

Once edema volume reaches a nadir, the maintenance approach to the limb will require the use of fitted elastic garments for use during non-recumbency; nocturnal compression may also be required. Relatively inelastic sleeves and underwear that transmit high-grade compression (40 to 80 mm Hg) will prevent reaccumulation of fluid after successful decongestive treatments.

Garments must be fitted properly and replaced every 3 to 6 months. Complete decongestive physiotherapy, including manual lymphatic drainage, compression bandaging, garments and skin care, is an effective treatment modality for many patients for chronic lymphatic dysfunction.

When examined in a series of patients with either upper or lower extremity lymphedema, with an average follow-up of 9 months, average volume reductions of 59% and 68% were observed in upper and lower limbs, respectively;14 maintenance self-management technique are effective in sustaining the majority of this benefit in compliant patients.14,15

Ancillary therapeutic measures can be considered as an adjunct to the elements of standard decongestive lymphatic therapy.10 Notably, intermittent pneumatic compression has been shown to add benefit to the decompressive effects of standard therapies, albeit in the context of cancer-associated, rather than primary, lymphedema.16 Other modalities, including low-level laser application17 and hyperthermia18 continue to be investigated.

Insight into the fundamental pathophysiology of lymphedema remains incomplete. It is clear, however, that the limb swelling that results from the increased volume of interstitial fluid and progressive fibrotic changes causes the patients to experience impairment in functions of daily living and engenders psychological dysfunction that stems from the chronic disease state and the associated impairment in body image and self-esteem.19–21

While a cure for lymphedema does not exist, intensive and sustained decongestive lymphatic physiotherapy should be advocated as the treatment choice to reduce edema, symptoms and functional impairment in patients with both simple and complex forms of lymphedema.


Saturday, February 04, 2006

Lymphatic microsurgery for the treatment of lymphedema.


Jan. 26, 2006

Campisi C, Davini D, Bellini C, Taddei G, Villa G, Fulcheri E, Zilli A, Da Rin E, Eretta C, Boccardo F.

Section of Lymphatic Surgery and Microsurgery, Department of Surgery, S. Martino Hospital, University of Genoa, Genoa, Italy.

One of the main problems of microsurgery for lymphedema consists of the discrepancy between the excellent technical possibilities and the subsequently insufficient reduction of the lymphoedematous tissue fibrosis and sclerosis. Appropriate treatment based on pathologic study and surgical outcome have not been adequately documented. Over the past 25 years, more than 1000 patients with peripheral lymphedema have been treated with microsurgical techniques.

Derivative lymphatic micro-vascular procedures has today its most exemplary application in multiple lymphatic-venous anastomoses (LVA). For those cases where a venous disease is associated to more or less latent or manifest lymphostatic pathology of such severity to contraindicate a lymphatic-venous shunt, reconstructive lymphatic microsurgery techniques have been developed (autologous venous grafts or lymphatic-venous-Iymphatic-plasty - LVLA).

Objective assessment was undertaken by water volumetry and lymphoscintigraphy. Subjective improvement was noted in 87% of patients. Objectively, volume changes showed a significant improvement in 83%, with an average reduction of 67% of the excess volume. Of those patients followed-up, 85% have been able to discontinue the use of conservative measures, with an average follow-up of more than 7 years and average reduction in excess volume of 69%. There was a 87% reduction in the incidence of cellulitis after microsurgery. Microsurgical lymphatic-venous anastomoses have a place in the treatment of peripheral lymphedema and should be the therapy of choice in patients who are not sufficiently responsive to nonsurgical treatment.

Improved results can be expected with operations performed earlier at the very first stages of lymphedema.

(c) 2006 Wiley-Liss, Inc.

Microsurgery 26: 65-69, 2006.PMID: 16444753

[PubMed - as supplied by publisher]

Is there a role for microsurgery in the prevention of arm lymphedema


January 26, 2006

Campisi C, Davini D, Bellini C, Taddei G, Villa G, Fulcheri E, Zilli A, Da Rin E, Eretta C, Boccardo F.

Section of Lymphatic Surgery and Microsurgery, Department of Surgery, S. Martino Hospital, University of Genoa, Genoa, Italy.

The secondary lymphedema of the upper limb (post-mastectomy lymphedema) has an incidence, in patients who underwent axillary lymphadenectomy for breast cancer, between 5 to 25%, up to 40% after radiotherapic treatment.

We studied 50 patients treated for breast cancer. The patients were divided in two groups of 25 each, comparable for age, sex, pathology and treatment and followed up to 5 years after operation for breast.

One group of 25 patients was controlled only clinically (physical examination, water volumetry) at 1-3-6 months and 1-3-5 years from breast cancer treatment.

The other group of 25 patients was followed also by lymphatic scintigraphy performed pre-operatively and after 1-3-6 months and 1- 3-5 years from operation. In the first group, followed only clinically, lymphedema appeared in 9 patients after a period variable from 1 week to 2 years, with highest incidence between 3 and 6 months. In the second group of 25 patients, the preventive therapeutic protocol allowed to have a clinically evident arm lymphedema only in 2 patients.

The comparison of the two groups of 25 patients proved a statistically significant difference in the appearance of arm secondary lymphedema (p = 0.01, using Fisher's exact test). The diagnostic and therapeutic preventive procedures allow to reduce the incidence rate of lymphedema significantly, in comparison with patients who did not undergo this protocol of prevention.

(c) 2006 Wiley-Liss, Inc. Microsurgery 26: 70-72, 2006. PMID: 16444710

Thursday, February 02, 2006

Aqua Lymphatic Therapy for Postsurgical Breast Cancer Lymphedema - Page Two

Patient 2: Initially, this participant's affected left arm volume was 2,556 ml compared with a volume of 2,522 ml in her healthy right arm. After 14 months in ALT, she achieved a volume of 2,440 ml, which is a reduction of 116 ml. She began the ALT program with 1% edema in her affected left arm as compared to her healthy right arm. After 14 months, she demonstrated a -5% edema in the affected left arm as compared to her healthy right arm and the lymphedema under her axillary area was smaller and softer. Subjectively, she reported that her arm was stronger and that she was more confident in using her arm in functional tasks than before the self-treatment program. Additionally, this participant went on 2 long vacations of 1-month duration, using air travel and although she forgot to take her compression garment, her lymphedema did not return.

Patient 3: Initially, this participant's left arm volume was 3,548 ml compared with a volume of 3,148 ml in her healthy right healthy arm. After 14 months in the ALT program, she achieved a volume of 3,222 ml, which is a 326 ml reduction. This participant began the program with a 13% increase in edema in her affected arm as compared to her healthy right arm. After 14 months, her edema in the affected arm decreased to 2% in comparison to the healthy arm. Additionally, the lymphedema under her axillary area disappeared.


The ALT protocol described in this article provides women in the maintenance phase of lymphedema with an effective and pleasurable method to promote adherence to self-treatment methods. Aquatic lymphatic therapy provides women with the following benefits: first, ALT uses the properties of water, specifically the buoyant force, hydrostatic pressure, water viscosity, and water temperature, to maintain or improve lymphedema reductions achieved during the intensive treatment phase with CDT. The ALT method may be effective because the hydrostatic pressure of water has the potential to remove the fluid and then the self-massage and exercise promote protein removal using healthy lymphotomes. Second, ALT promotes self-efficacy by educating the participants to use the sequence and the slow rhythm of appropriate exercises so the participants can take responsibility for performing their individualized protocol.

Third, ALT provides women with a support group with all of its advantages.16 Fourth, this program includes monitoring by a physical therapist to address changes on an individualized basis, and the use of feedback charts to empower a woman to monitor her status and to develop appropriate self-treatment strategies. Finally, there is active self treatment as the participants use their muscles throughout the entire session, in contrast to conventional treatments using passive techniques.

The ALT was used in these cases as an effective tool during the maintenance phase of lymphedema when a woman is responsible for treating herself. The maintenance phase also may consist of daily compression with a low-stretch elastic sleeve, skin care, continued 'remedial' exercises, and repeated light massage as needed.7

All 3 women demonstrated good adherence with the ALT program. Two of the participants (Participant 2 and 3) were considered nonadherent with conventional maintenance phase treatments, since they did not wear their compression garments or perform the self massage and exercises. However, adherence to this method of maintenance was high for both of these participants.

Lastly, the summer temperature in Israel can reach up to 40°C with heat waves even in April and May. In spite of the changes in the climate through the 14 months, the amplitude of changes in the volume of the lymphedematous limbs were not acute. During April 2003 there were no sessions due to pool reconstruction, but as seen in Figures 10 and 11, the effect on the patients' conditions was a brief one and didn't last.


These case reports demonstrate the benefits that are typically derived from ALT by women in the maintenance phase of lymphedema treatment. During the 14 months of ALT none of these participants experienced exacerbations of their lymphedema and all of these women demonstrated further reductions in the volumes of their affected arm. These 3 women represent typical types of women that participate in the ALT program.

Participant 1 represents women who develop lymphedema even after receiving proper care and exhibiting good compliance to traditional maintenance techniques. Of interest was the fact that participant 1 experienced further marked improvements in her lymphedema with ALT treatments, as traditional techniques did not appear to adequately control her condition.

Additionally, women like participant 1 who develop lymphedema despite good care typically are required to wear a compression sleeves between self-treatment sessions as this participant was required to do.

Participant 2 represents women who achieve 100% reductions of their lymphedema after the initial intensive phase of the conventional treatment, or come for self-treatment to prevent the exacerbations of their lymphedema. Women like participant 2 are able to maintain good results with no further self treatment between sessions.

Participant 3 represents women who have lymphedema but struggle to adhere to traditional maintenance techniques. Participant 3 demonstrated good adherence to ALT and was able to show good improvements in her condition. This participant did not require a compression sleeve between sessions, like many women in the maintenance phase. For women similar to participant 3, ALT may be an additional tool in their maintenance phase for controlling lymphedema.

Further studies are needed to answer the following questions:

* What are the long-term and short-term physiological effects behind the ALT?
* Does ALT activate healthy lymphotomes or collateral vessels to take over the work for affected lymphotomes?
* How do the results of ALT compare to traditional land-based maintenance programs for lymphedema in terms of preventing exacerbations or further reducing lymphedema volumes?
* What are the differences in adherence between traditional maintenance protocols and ALT?
* Can this method be a successful alternative to the conventional methods for arm and leg lymphedema in places where conventional treatment is not available due to cost or availability or trained health care practitioners?
* What is the actual cost comparison between traditional maintenance protocols and ALT?
* Can ALT be used between phases one and two in order to improve outcomes in cases where a 100% reduction was not achieved by the CLT/CDT?

The ALT program has been successfully used at our facility for more than 2 years. We are continuing to gather additional data on the effects of aqua lymphatic therapy on patients.

However, further clinical research is required to modify, refine, and provide evidence for the utility of this approach in other groups of patients including those with lower extremity lymphedemas or lymphedema that is not the sequela of cancer treatments.


We express our deepest gratitude to Prof Judith R. Casley-Smith, Chairman of the Lymphoedema Association of Australia, for her support in the development of the Aqua Lymphatic Therapy; and for Dr Anna Towers, Director, Palliative Care Division, McGill Department of Oncology; and Rachel Pritzker, President of the Lymphedema Association of Quebec (AQL/LAQ) for their helpful comments on an earlier draft of the manuscript.

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Dorit Tidhar, BPT, Aqua Lymphatic Therapist, Hydrotherapeutic Center of Shaar-Hanegev, Israel
Avi Shimony, MD, Soroka University Medical Center, Israel
Jacqueline Drouin, PT, PhD, Assistant Professor, Physical Therapy Department, The University of Michigan-Flint, USA
Copyright Rehabilitation in Oncology 2004 Provided by ProQuest Information and Learning Company. All rights Reserved