Clearance of interstitial fluid (ISF) and CSF (CLIC) group-part of Vascular Professional Interest Area (PIA), updates in 2022-2023. Cerebrovascular disease and the failure of elimination of Amyloid-ß from the brain and retina with age and Alzheimer's disease: Opportunities for therapy.

This editorial summarizes advances from the Clearance of Interstitial Fluid and Cerebrospinal Fluid (CLIC) group, within the Vascular Professional Interest Area (PIA) of the Alzheimer's Association International Society to Advance Alzheimer's Research and Treatment (ISTAART). The overarching objectives of the CLIC group are to: (1) understand the age-related physiology changes that underlie impaired clearance of interstitial fluid (ISF) and cerebrospinal fluid (CSF) (CLIC); (2) understand the cellular and molecular mechanisms underlying intramural periarterial drainage (IPAD) in the brain; (3) establish novel diagnostic tests for Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), retinal amyloid vasculopathy, amyloid-related imaging abnormalities (ARIA) of spontaneous and iatrogenic CAA-related inflammation (CAA-ri), and vasomotion; and (4) establish novel therapies that facilitate IPAD to eliminate amyloid ß (Aß) from the aging brain and retina, to prevent or reduce AD and CAA pathology and ARIA side events associated with AD immunotherapy.

Central nervous system zoning: How brain barriers establish subdivisions for CNS immune privilege and immune surveillance.

The central nervous system (CNS) coordinates all our body functions. Neurons in the CNS parenchyma achieve this computational task by high speed communication via electrical and chemical signals and thus rely on a strictly regulated homeostatic environment, which does not tolerate uncontrolled entry of blood components including immune cells. The CNS thus has a unique relationship with the immune system known as CNS immune privilege. Previously ascribed to the presence of blood-brain barriers and the lack of lymphatic vessels in the CNS parenchyma prohibiting, respectively, efferent and afferent connections with the peripheral immune system, it is now appreciated that CNS immune surveillance is ensured by cellular and acellular brain barriers that limit immune cell and mediator accessibility to specific compartments at the borders of the CNS. CNS immune privilege is established by a brain barriers anatomy resembling the architecture of a medieval castle surrounded by two walls bordering a castle moat. Built for protection and defense this two-walled rampart at the outer perimeter of the CNS parenchyma allows for accommodation of different immune cell subsets and efficient monitoring of potential danger signals derived from inside or outside of the CNS parenchyma. It enables effective mounting of immune responses within the subarachnoid or perivascular spaces, while leaving the CNS parenchyma relatively undisturbed. In this study, we propose that CNS immune privilege rests on the proper function of the brain barriers, which allow for CNS immune surveillance but prohibit activation of immune responses from the CNS parenchyma unless it is directly injured.

Cerebrospinal fluid outflow: a review of the historical and contemporary evidence for arachnoid villi, perineural routes, and dural lymphatics.

Cerebrospinal fluid (CSF) is produced by the choroid plexuses within the ventricles of the brain and circulates through the subarachnoid space of the skull and spinal column to provide buoyancy to and maintain fluid homeostasis of the brain and spinal cord. The question of how CSF drains from the subarachnoid space has long puzzled scientists and clinicians. For many decades, it was believed that arachnoid villi or granulations, outcroppings of arachnoid tissue that project into the dural venous sinuses, served as the major outflow route. However, this concept has been increasingly challenged in recent years, as physiological and imaging evidence from several species has accumulated showing that tracers injected into the CSF can instead be found within lymphatic vessels draining from the cranium and spine. With the recent high-profile rediscovery of meningeal lymphatic vessels located in the dura mater, another debate has emerged regarding the exact anatomical pathway(s) for CSF to reach the lymphatic system, with one side favoring direct efflux to the dural lymphatic vessels within the skull and spinal column and another side advocating for pathways along exiting cranial and spinal nerves. In this review, a summary of the historical and contemporary evidence for the different outflow pathways will be presented, allowing the reader to gain further perspective on the recent advances in the field. An improved understanding of this fundamental physiological process may lead to novel therapeutic approaches for a wide range of neurological conditions, including hydrocephalus, neurodegeneration and multiple sclerosis.

Lymphatic MAFB regulates vascular patterning during developmental and pathological lymphangiogenesis.

MAFB is a transcription factor involved in the terminal differentiation of several cell types, including macrophages and keratinocytes. MAFB is also expressed in lymphatic endothelial cells (LECs) and is upregulated by VEGF-C/VEGFR-3 signaling. Recent studies have revealed that MAFB regulates several genes involved in lymphatic differentiation and that global Mafb knockout mice show defects in patterning of lymphatic vessels during embryogenesis. However, it has remained unknown whether this effect is LEC-intrinsic and whether MAFB might also be involved in postnatal lymphangiogenesis. We established conditional, lymphatic-specific Mafb knockout mice and found comparable lymphatic patterning defects during embryogenesis as in the global MAFB knockout. Lymphatic MAFB deficiency resulted in increased lymphatic branching in the diaphragm at P7, but had no major effect on lymphatic patterning or function in healthy adult mice. By contrast, tumor-induced lymphangiogenesis was enhanced in mice lacking lymphatic MAFB. Together, these data reveal that LEC-expressed MAFB is involved in lymphatic vascular morphogenesis during embryonic and postnatal development as well as in pathological conditions. Therefore, MAFB could represent a target for therapeutic modulation of lymphangiogenesis.

Differential effects of anaesthesia on the contractility of lymphatic vessels in vivo.

KEY POINTS: Contractility of lymphatic collectors is essential for the functionality of the lymphatic system and, thus, for lymph flow. Previously published rates of lymphatic collectors in mice vary from 1.1 to 17 contractions/min with little agreement between investigators. In this study, we focused on the effects of different anaesthesia regimens on lymphatic vessel contractility using in vivo imaging approaches. We show that isoflurane and pentobarbital have an inhibitory effect on lymphatic contractility compared to mice under other anaesthesia regimens and in awake conditions. These results should help to establish a standardization of lymphatic contraction studies in mice and may also have relevance for patients undergoing anaesthesia during surgery. ABSTRACT: Contractions of collecting lymphatic vessels are essential for the function of the lymphatic vascular system, due to the lack of a central pump to drive flow. A wide range of physiological contraction frequencies and strengths have been reported in previous in vivo studies in mice. This is probably due to the different types of anaesthesia that have been used and which might have exerted direct influences on lymphatic vessel function. We investigated six commonly used anaesthesia regimens for their influence on lymphatic vessel contractility using near-infrared in vivo imaging approaches. Non-invasive imaging of the lymphatic leg collector revealed distinct effects of the anaesthesia regimens with reduced contraction activity under isoflurane and pentobarbital anaesthesia. Isoflurane also reduced the contractility of near-infrared dye-loaded vessels during invasive imaging of the lymphatic flank collector whereas the combination of ketamine/xylazine/acepromazine had no major effects. The transport time of a lymphatic-specific dye from the skin through the lymphatic vasculature to the systemic bloodstream was also delayed under isoflurane anaesthesia. Based on these results, we recommend use of combinations of ketamine and medetomidine for future non-invasive studies and of ketamine, xylazine and acepromazine for invasive studies. Beyond their importance for facilitating the interpretation and planning of animal studies, our findings might also have relevance for human patients undergoing anaesthesia for surgical procedures.

Transcriptional profiling of breast cancer-associated lymphatic vessels reveals VCAM-1 as regulator of lymphatic invasion and permeability.

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin α4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis.

Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells.

Vascular endothelial growth factor (VEGF)-A is a well-known major chemoattractant driver of angiogenesis--the formation of new blood vessels from pre-existing ones. However, the repellent factors that fine-tune this angiogenic process remain poorly characterized. We investigated the expression and functional role of endothelial cell-derived semaphorin 3A (Sema3A) in retinal angiogenesis, using genetic mouse models. We found Sema3a mRNA expression in the ganglion cell layer and the presence of Sema3A protein on larger blood vessels and at the growing front of blood vessels in neonatal retinas. The Sema3A receptors neuropilin-1 and plexin-A1 were expressed by retinal blood vessels. To study the endothelial cell-specific role of Sema3A, we generated endothelial cell-specific Sema3A knockout mouse strains by constitutive or inducible vascular endothelial cadherin-Cre-mediated gene disruption. We found that in neonatal retinas of these mice, both the number and the length of tip cell filopodia were significantly increased and the leading edge growth pattern was irregular. Retinal explant experiments showed that recombinant Sema3A significantly decreased VEGF-A-induced filopodia formation. Endothelial cell-specific knockout of Sema3A had no impact on blood vessel density or skin vascular leakage in adult mice. These findings indicate that endothelial cell-derived Sema3A exerts repelling functions on VEGF-A-induced tip cell filopodia and that a lack of this signaling cannot be rescued by paracrine sources of Sema3A.

Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain.

The relationships between cerebrospinal fluid (CSF) and brain interstitial fluid are still being elucidated. It has been proposed that CSF within the subarachnoid space will enter paravascular spaces along arteries to flush through the parenchyma of the brain. However, CSF also directly exits the subarachnoid space through the cribriform plate and other perineural routes to reach the lymphatic system. In this study, we aimed to elucidate the functional relationship between CSF efflux through lymphatics and the potential influx into the brain by assessment of the distribution of CSF-infused tracers in awake and anesthetized mice. Using near-infrared fluorescence imaging, we showed that tracers quickly exited the subarachnoid space by transport through the lymphatic system to the systemic circulation in awake mice, significantly limiting their spread to the paravascular spaces of the brain. Magnetic resonance imaging and fluorescence microscopy through the skull under anesthetized conditions indicated that tracers remained confined to paravascular spaces on the surface of the brain. Immediately after death, a substantial influx of tracers occurred along paravascular spaces extending into the brain parenchyma. We conclude that under normal conditions a rapid CSF turnover through lymphatics precludes significant bulk flow into the brain.

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

Prominent Lymphatic Vessel Hyperplasia with Progressive Dysfunction and Distinct Immune Cell Infiltration in Lymphedema.

Lymphedema is a common complication that occurs after breast cancer treatment in up to 30% of the patients undergoing surgical lymph node excision. It is associated with tissue swelling, fibrosis, increased risk of infection, and impaired wound healing. Despite the pronounced clinical manifestations of the disease, little is known about the morphological and functional characteristics of the lymphatic vasculature during the course of lymphedema progression. We used an experimental murine tail lymphedema model where sustained fluid stasis was generated on disruption of lymphatic flow, resulting in chronic edema formation with fibrosis and adipose tissue deposition. Morphological analysis of the lymphatic vessels revealed a dramatic expansion during the course of the disease, with active proliferation of lymphatic endothelial cells at the early stages of lymphedema. The lymphatic capillaries exhibited progressively impaired tracer filling and retrograde flow near the surgery site, whereas the collecting lymphatic vessels showed a gradually decreasing contraction amplitude with unchanged contraction frequency, leading to lymphatic contraction arrest at the later stages of the disease. Lymphedema onset was associated with pronounced infiltration by immune cells, predominantly Ly6G(+) and CD4(+) cells, which have been linked to impaired lymphatic vessel function.

High-Fat Diet in the Absence of Obesity Does Not Aggravate Surgically Induced Lymphoedema in Mice.

BACKGROUND: Lymphoedema represents the cardinal manifestation of lymphatic dysfunction and is associated with expansion of the adipose tissue in the affected limb. In mice, high-fat diet (HFD)-induced obesity was associated with impaired collecting lymphatic vessel function, and adiposity aggravated surgery-induced lymphoedema in a mouse model. The aim of the current study was to investigate whether adiposity is necessary to impair lymphatic function or whether increased lipid exposure alone might be sufficient in a surgical lymphoedema model. METHODS: To investigate the role of increased lipid exposure in lymphoedema development we used a well-established mouse tail lymphoedema model. Female mice were subjected to a short-term (6 weeks) HFD, without development of obesity, before surgical induction of lymphedema. Lymphoedema was followed over a period of 6 weeks measuring oedema, evaluating tissue histology and lymphatic vascular function. RESULTS: HFD increased baseline angiogenesis and average lymphatic vessel size in comparison to the chow control group. Upon induction of lymphedema, HFD-treated mice did not exhibit aggravated oedema and no morphological differences were observed in the blood and lymphatic vasculature. Importantly, the levels of fibro-adipose tissue deposition were comparable between the 2 groups and lymphatic vessel function was not impaired as a result of the HFD. Although the net immune cell infiltration was comparable, the HFD group displayed an increased infiltration of macrophages, which exhibited an M2 polarization phenotype. CONCLUSIONS: These results indicate that increased adiposity rather than dietary influences determines predisposition to or severity of lymphedema.

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Microneedles for the Noninvasive Structural and Functional Assessment of Dermal Lymphatic Vessels.

The medical and scientific communities' interest in the lymphatic system has been growing rapidly in recent years. It has become evident that the lymphatic system is much more than simply a homeostasis controller and that it plays key roles in several pathological conditions. This work describes the identification of the optimal combination of poly(N-vinylpyrrolidone) and a near-infrared dye (indocyanine green) for the manufacturing of soluble microneedles and their application to the imaging of the lymphatic system. Upon application to the skin, the microneedle-bearing indocyanine green is delivered in the dermal layer, where the lymphatic vessels are abundant. The draining lymphatics can then be visualized and the clearance kinetics from the administration site simply determined using a near-infrared camera. This painless functional "tattooing" procedure can be used for quantitative assessment of the dermal lymphatic function in several dermal conditions and treatment-response evaluations. The two components of these microneedles are extensively used in routine medical care, potentially leading to rapid clinical translation. Moreover, this procedure may have a significant impact on preclinical lymphatic studies.

Decline of lymphatic vessel density and function in murine skin during aging.

Lymphatic vessels play important roles in the pathogenesis of many conditions that have an increased prevalence in the elderly population. However, the effects of the aging process on the lymphatic system are still relatively unknown. We have applied non-invasive imaging and whole-mount staining techniques to assess the lymphatic vessel function and morphology in three different age groups of mice: 2 months (young), 7 months (middle-aged), and 18 months (aged). We first developed and validated a new method to quantify lymphatic clearance from mouse ear skin, using a lymphatic-specific near-infrared tracer. Using this method, we found that there is a prominent decrease in lymphatic vessel function during aging since the lymphatic clearance was significantly delayed in aged mice. This loss of function correlated with a decreased lymphatic vessel density and a reduced lymphatic network complexity in the skin of aged mice as compared to younger controls. The blood vascular leakage in the skin was slightly increased in the aged mice, indicating that the decreased lymphatic function was not caused by a reduced capillary filtration in aged skin. The decreased function of lymphatic vessels with aging might have implications for the pathogenesis of a number of aging-related diseases.

Interleukin-7 is produced by afferent lymphatic vessels and supports lymphatic drainage.

The cytokine interleukin (IL)-7 exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7, but also express the IL-7 receptor chains IL-7Rα and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. Whereas in IL-7Rα(-/-) mice, dermal lymphatic vessels (LVs) were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Rα(-/-) bone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Rα expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Rα(-/-) mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point toward an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage.

Molecular mechanisms and imaging of lymphatic metastasis.

In many types of cancer, tumors metastasize through the lymphatic system to draining lymph nodes. These sentinel lymph nodes have gained increased attention as a prognostic indicator for the severity of the disease, leading to the sentinel lymph node mapping and biopsy procedure to be accepted as standard-of-care for breast cancer and melanoma. However, many limitations exist with this procedure resulting in high false negative rates. In this review we highlight the new advances in the understanding of the molecular mechanisms of lymphangiogenesis and tumor metastasis that may lead to improved strategies in the detection of the sentinel lymph nodes and therapeutic interventions to prevent further tumor spread. In addition, advances in imaging technology are allowing new approaches for anatomical mapping of lymphatic drainage patterns and molecular imaging strategies that may improve detection of metastatic tumor cells within sentinel lymph nodes.

VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis.

The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2 Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

Non-invasive dynamic near-infrared imaging and quantification of vascular leakage in vivo.

Preclinical vascular research has been hindered by a lack of methods that can sensitively image and quantify vascular perfusion and leakage in vivo. In this study, we have developed dynamic near-infrared imaging methods to repeatedly visualize and quantify vascular leakage in mouse skin in vivo, and we have applied these methods to transgenic mice with overexpression of vascular endothelial growth factors VEGF-A or -C. Near-infrared dye conjugates were developed to identify a suitable vascular tracer that had a prolonged circulation lifetime and slow leakage into normal tissue after intravenous injection. Dynamic simultaneous imaging of ear skin and a large blood vessel in the leg enabled determination of the intravascular signal (blood volume fraction) from the tissue signal shortly after injection and quantifications of vascular leakage into the extravascular tissue over time. This method allowed for the sensitive detection of increased blood vascularity and leakage rates in K14-VEGF-A transgenic mice and also reliably measured inflammation-induced changes of vascularity and leakage over time in the same mice. Measurements after injection of recombinant VEGF-A surprisingly revealed increased blood vascular leakage and lymphatic clearance in K14-VEGF-C transgenic mice which have an expanded cutaneous lymphatic vessel network, potentially indicating unanticipated effects of lymphatic drainage on vascular leakage. Increased vascular leakage was also detected in subcutaneous tumors, confirming that the method can also be applied to deeper tissues. This new imaging method might facilitate longitudinal investigations of the in vivo effects of drug candidates, including angiogenesis inhibitors, in preclinical disease models.

Dynamics of lymphatic regeneration and flow patterns after lymph node dissection.

Knowledge about the mechanisms of regeneration of the lymphatic vasculature after surgical trauma is essential for the development of strategies for the prevention and therapy of lymphedema. However, little is known about the alterations of lymphatic flow directly after surgical trauma. We investigated lymphatic function in mice using near-infrared imaging for a period of 4 weeks after surgeries that mimic sentinel lymph node biopsy (SLNB) or axillary lymph node dissection (ALND), by removal of the popliteal lymph node (LN) alone or together with the popliteal fat pad, respectively. SLNB-like surgery did not cause changes in lymphatic drainage in the majority of cases. In contrast, lymphatic drainage impairment shown by collecting vessel rupture, dermal backflow and rerouting of lymph flow via collateral vessels were observed after ALND-like surgery. All collateral vessels drained to the inguinal LN. These results indicate that less invasive surgery prevents lymphatic decompensation. They also reveal the development and maturation of collateral lymphatic vessels after extensive surgical trauma, which reroute the flow of lymph towards a different LN. These findings might be helpful for the development of strategies to prevent and/or treat post-surgical lymphedema.