Spatio-Temporal Changes of Lymphatic Contractility and Drainage Patterns following Lymphadenectomy in Mice

Objective

To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis.

Background

Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis.

Methods

Changes in lymphatic “pumping” function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis.

Results

NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy.

Conclusions

Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage.     

MR Lymphography of Lymphatic Vessels in Lower Extremity with Gynecologic Oncology-Related Lymphedema

Objective

To characterize lymphatic vessel morphology in lower extremity lymphedema using MR lymphography at 3T.

Study Design

Forty females with lower extremity lymphedema secondary to gynecologic carcinoma treatment underwent MR lymphography (MRL) at 3T. Lymphatic vessel morphology in normal and affected limbs was compared.

Results

The median diameter of the lymphatic vessels in swollen calf and thigh were significantly larger than that in the contralateral calf and thigh, respectively (p<0.05). The median number of lymphatic vessels visualized in normal calf was less than that in the lymphedematous calf (p<0.01), while no significant difference was found between the normal thigh and swollen thigh. Lymphatic vessel number in the affected calf was significantly greater than that in affected thigh and the mean diameter of affected calf was also significantly wider than that of affected thigh (p<0.01). Mean diameter of lymphatic vessels in the affected calf was significantly different between stage I and stage III (p<0.05), but not significantly different between stages I and II, and between stages II and III (p>0.05). The median number of lymphatic vessels for affected calf showed significant difference between stage I and stage III, and between stage II and stage III (p<0.05), but no significant difference between stage I and stage II (p>0.05). There was no significant difference in mean diameter or median number of lymphatic vessels in the affected thigh found between different stages (p>0.05).

Conclusion

There are significant differences in the number or diameter of lymphatic vessels between normal and affected limbs and there are significant differences for affected calf between early and late stages of lymphedema; therefore, MR lymphography can be helpful in diagnosis or clinical staging for lower extremity with gynecologic oncology-related lymphedema.     

Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation

Background

Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/Principal Findings

B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A₂ (PLA₂) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA₂ homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/Significance

Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA₂s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.     

Mechanisms of lymphatic regeneration after tissue transfer

Introduction

Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.

Methods

Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.

Results

Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.

Conclusions

Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.     

Lymphatic Territories (Lymphosomes) in a Canine: An Animal Model for Investigation of Postoperative Lymphatic Alterations

Background

Lymph node dissection is often performed as a part of surgical treatment for breast cancer and malignant melanoma to prevent malignant cells from traveling via the lymphatic system. Currently little is known about postoperative lymphatic drainage pattern alterations. This knowledge may be useful for management of recurrent cancer and prevention of breast cancer related lymphedema. We mapped the complete superficial lymphatic system of a dog and used this canine model to perform preliminary studies of lymphatic architectural changes in postoperative condition.

Methods

Lymphatic territories (lymphosomes) were mapped with 4 female mongrel carcasses using an indocyanine green (ICG) fluorescent lymphography and a radiographic microinjection technique. Two live dogs were then subjected to unilateral lymph node dissection of lymph basins of the forelimb, and ICG lymphography and lymphangiogram were performed 6 months after the surgery to investigate lymphatic changes. Lymphatic patterns in the carcass were then compared with postoperative lymphatic patterns in the live dogs.

Results

Ten lymphosomes were identified, corresponding with ten lymphatic basins. Postoperative fluorescent lymphographic images and lymphangiograms in the live dogs revealed small caliber lymphatic network fulfilling gaps in the surgical area and collateral lymphatic vessels arising from the network connecting to lymph nodes in the contralateral and ipsilateral neck in one dog and the ipsilateral subclavicular vein in another dog.

Conclusion

Our canine lymphosome map allowed us to observe lymphatic collateral formations after lymph node dissection in live dogs. This canine model may help clarify our understanding of postoperative lymphatic changes in humans in future studies.     

Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels

Background

The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.

Methodology/Principal Findings

We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.

Conclusions

To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.     

CD4+ Cells Regulate Fibrosis and Lymphangiogenesis in Response to Lymphatic Fluid Stasis

Introduction

Lymphedema is a chronic disorder that occurs commonly after lymph node removal for cancer treatment and is characterized by swelling, fibrosis, inflammation, and adipose deposition. Although previous histological studies have investigated inflammatory changes that occur in lymphedema, the precise cellular make up of the inflammatory infiltrate remains unknown. It is also unclear if this inflammatory response plays a causal role in the pathology of lymphedema. The purpose of this study was therefore to characterize the inflammatory response to lymphatic stasis and determine if these responses are necessary for the pathological changes that occur in lymphedema.

Methods

We used mouse-tail lymphedema and axillary lymph node dissection (ANLD) models in order to study tissue inflammatory changes. Single cell suspensions were created and analyzed using multi-color flow cytometry to identify individual cell types. We utilized antibody depletion techniques to analyze the causal role of CD4+, CD8+, and CD25+ cells in the regulation of inflammation, fibrosis, adipose deposition, and lymphangiogenesis.

Results

Lymphedema in the mouse-tail resulted in a mixed inflammatory cell response with significant increases in T-helper, T-regulatory, neutrophils, macrophages, and dendritic cell populations. Interestingly, we found that ALND resulted in significant increases in T-helper cells suggesting that these adaptive immune responses precede changes in macrophage and dendritic cell infiltration. In support of this we found that depletion of CD4+, but not CD8 or CD25+ cells, significantly decreased tail lymphedema, inflammation, fibrosis, and adipose deposition. In addition, depletion of CD4+ cells significantly increased lymphangiogenesis both in our tail model and also in an inflammatory lymphangiogenesis model.

Conclusions

Lymphedema and lymphatic stasis result in CD4+ cell inflammation and infiltration of mature T-helper cells. Loss of CD4+ but not CD8+ or CD25+ cell inflammation markedly decreases the pathological changes associated with lymphedema. In addition, CD4+ cells regulate lymphangiogenesis during wound repair and inflammatory lymphangiogenesis.     

Anti-Inflammatory Pharmacotherapy with Ketoprofen Ameliorates Experimental Lymphatic Vascular Insufficiency in Mice

Background

Disruption of the lymphatic vasculature causes edema, inflammation, and end-tissue destruction. To assess the therapeutic efficacy of systemic anti-inflammatory therapy in this disease, we examined the impact of a nonsteroidal anti-inflammatory drug (NSAID), ketoprofen, and of a soluble TNF-α receptor (sTNF-R1) upon tumor necrosis factor (TNF)-α activity in a mouse model of acquired lymphedema.

Methods and Findings

Lymphedema was induced by microsurgical ablation of major lymphatic conduits in the murine tail. Untreated control mice with lymphedema developed significant edema and extensive histopathological inflammation compared to sham surgical controls. Short-term ketoprofen treatment reduced tail edema and normalized the histopathology while paradoxically increasing TNF-α gene expression and cytokine levels. Conversely, sTNF-R1 treatment increased tail volume, exacerbated the histopathology, and decreased TNF-α gene expression. Expression of vascular endothelial growth factor-C (VEGF-C), which stimulates lymphangiogenesis, closely correlated with TNF-α expression.

Conclusions

Ketoprofen therapy reduces experimental post-surgical lymphedema, yet direct TNF-α inhibition does not. Reducing inflammation while preserving TNF-α activity appears to optimize the repair response. It is possible that the observed favorable responses, at least in part, are mediated through enhanced VEGF-C signaling.     

Side-to-End Lymphaticovenular Anastomosis through Temporary Lymphatic Expansion

Objective

The number of bypasses is the most important factor in lymphaticovenular anastomosis (LVA) for lymphedema treatment. Side-to-end (S-E) LVA, which can bypass bidirectional lymph flows via one anastomosis, is considered to be the most efficient bypass, but creation of lateral window to a small lymphatic vessel is technically demanding. To overcome the difficulty, we introduced S-E anastomosis through temporary lymphatic expansion (SEATTLE) procedure in S-E LVA.

Methods

This was a retrospective observational study set in a teaching hospital. Forty eight lower extremity lymphedema (LEL) patients underwent LVA. S-E LVAs were performed with (SEATTLE group) or without (non-SEATTLE group) temporary lymphatic expansion. S-E LVAs were evaluated to compare anastomosis result in SEATTLE and non-SEATTLE groups.

Results

S-E LVAs resulted in 44 anastomoses in SEATTLE group (n = 25) and 37 anastomoses in non-SEATTLE group (n = 23). LEL index reduction in SEATTLE group was significantly greater than that in non-SEATTLE group (16.5±14.5 vs. 10.9±11.8, P = 0.041). Success rate of S-E LVA in SEATTLE group was significantly higher than that in non-SEATTLE group (95.5% vs 81.1%, P = 0.040). Thirty seven of 44 (84.1%) lymph vessels in SEATTLE group were successfully dilated by temporary lymphatic expansion maneuver. All of 9 failed S-E LVAs used a lymphatic vessel with diameter of 0.35 mm or smaller.

Conclusions

The SEATTLE procedure facilitates S-E LVA by a simple and easy maneuver. When the diameter of the lymphatic vessel is 0.35 mm or smaller even after the temporary lymphatic expansion maneuver, S-E LVA is not recommended due to relatively high failure rate.     

Hematopoietic Stem Cells Contribute to Lymphatic Endothelium

Background

Although the lymphatic system arises as an extension of venous vessels in the embryo, little is known about the role of circulating progenitors in the maintenance or development of lymphatic endothelium. Here, we investigated whether hematopoietic stem cells (HSCs) have the potential to give rise to lymphatic endothelial cells (LEC).

Methodology/Principal Findings

Following the transfer of marked HSCs into irradiated recipients, donor-derived LEC that co-express the lymphatic endothelial markers Lyve-1 and VEGFR-3 were identified in several tissues. HSC-derived LEC persisted for more than 12 months and contributed to ∼3–4% of lymphatic vessels. Donor-derived LECs were not detected in mice transplanted with common myeloid progenitors and granulocyte/macrophage progenitors, suggesting that myeloid lineage commitment is not a requisite step in HSC contribution to lymphatic endothelium. Analysis of parabiotic mice revealed direct evidence for the existence of functional, circulating lymphatic progenitors in the absence of acute injury. Furthermore, the transplantation of HSCs into Apc^Min/+ mice resulted in the incorporation of donor-derived LEC into the lymphatic vessels of spontaneously arising intestinal tumors.

Conclusions/Significance

Our results indicate that HSCs can contribute to normal and tumor associated lymphatic endothelium. These findings suggest that the modification of HSCs may be a novel approach for targeting tumor metastasis and attenuating diseases of the lymphatic system.     

Feasibility and Effectiveness of Basic Lymphedema Management in Leogane,Haiti, an Area Endemic for Bancroftian Filariasis

Background

Approximately 14 million persons living in areas endemic for lymphatic filariasis have lymphedema of the leg. Clinical studies indicate that repeated episodes of bacterial acute dermatolymphangioadenitis (ADLA) lead to progression of lymphedema and that basic lymphedema management, which emphasizes hygiene, skin care, exercise, and leg elevation, can reduce ADLA frequency. However, few studies have prospectively evaluated the effectiveness of basic lymphedema management or assessed the role of compressive bandaging for lymphedema in resource-poor settings.

Methodology/Principal Findings

Between 1995 and 1998, we prospectively monitored ADLA incidence and leg volume in 175 persons with lymphedema of the leg who enrolled in a lymphedema clinic in Leogane, Haiti, an area endemic for Wuchereria bancrofti. During the first phase of the study, when a major focus of the program was to reduce leg volume using compression bandages, ADLA incidence was 1.56 episodes per person-year. After March 1997, when hygiene and skin care were systematically emphasized and bandaging discouraged, ADLA incidence decreased to 0.48 episodes per person-year (P<0.0001). ADLA incidence was significantly associated with leg volume, stage of lymphedema, illiteracy, and use of compression bandages. Leg volume decreased in 78% of patients; over the entire study period, this reduction was statistically significant only for legs with stage 2 lymphedema (P = 0.01).

Conclusions/Significance

Basic lymphedema management, which emphasized hygiene and self-care, was associated with a 69% reduction in ADLA incidence. Use of compression bandages in this setting was associated with an increased risk of ADLA. Basic lymphedema management is feasible and effective in resource-limited areas that are endemic for lymphatic filariasis.     

Differentiation potential of CD14+ monocytes into myofibroblasts in patients with systemic sclerosis

Background

Circulating monocytes are a highly plastic and functionally heterogeneic cell type with an activated phenotype in patients with systemic sclerosis (SSc). CD14⁺ monocytes have the potential to differentiate into extra-cellular matrix (ECM) producing cells, possibly participating in fibrogenesis.

Aim

To study the effect of GM-CSF, IL-4 and endothelin -1 (ET-1) alone or in combination on monocyte differentiation into myofibroblasts.

Methods

CD14⁺ cells were isolated from peripheral blood from 14 SSc patients and healthy controls by positive selection and incubated with different combinations of GM-CSF, IL-4 and ET-1 for 14 days. Type-1 collagen and α-SMA were detected by Western blot, qPCR and confocal microscopy. HLA-DR, CD11c and CD14 expression was analysed by flow cytometry. A collagen gel contraction assay was performed for functional myofibroblast assessment.

Results

GM-CSF both induced collagen and α-SMA expression after 14 days. ET-1 further increased GM-CSF-induced collagen expression in a dose dependent manner up to 30-fold. IL-4/GM-CSF combination leads to a more DC-like phenotype of monocytes associated with reduced collagen and α-SMA expression compared to GM-CSF alone. Collagen and α-SMA expression was higher in monocytes from SSc patients and monocytes were more prone to obtain a spindle form. In contrast to controls, ET-1 and IL-4 alone were sufficient to induce α-SMA expression in monocytes from SSc patients. Despite the induction of α-SMA expression, monocyte-derived myofibroblasts only had a moderate capability of contraction in functional analyses.

Conclusion

SSc monocytes display increased maturation towards myofibroblasts demonstrated by their phenotype and α-SMA expression when compared to monocytes from healthy controls, however only with minor functional contraction properties.     

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy

Aims

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds).

Materials and Methods

Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy).

Results

Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections.

Conclusions

The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.     

Development,Alteration and Real Time Dynamics of Conjunctiva-Associated Lymphoid Tissue

Purpose

Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model.

Methods

Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy.

Results

Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min.

Conclusions

CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.     

Imaging Lymphatic System in Breast Cancer Patients with Magnetic Resonance Lymphangiography

Objective

To investigate the feasibility of gadolinium (Gd) contrast-enhanced magnetic resonance lymphangiography (MRL) in breast cancer patients within a typical clinical setting, and to establish a Gd-MRL protocol and identify potential MRL biomarkers for differentiating metastatic from non-metastatic lymph nodes.

Materials and Methods

32 patients with unilateral breast cancer were enrolled and divided into 4 groups of 8 patients. Groups I, II, and III received 1.0, 0.5, and 0.3 ml of intradermal contrast; group IV received two 0.5 ml doses of intradermal contrast. MRL images were acquired on a 3.0 T system and evaluated independently by two radiologists for the number and size of enhancing lymph nodes, lymph node contrast uptake kinetics, lymph vessel size, and contrast enhancement patterns within lymph nodes.

Results

Group III patients had a statistically significant decrease in the total number of enhancing axillary lymph nodes and lymphatic vessels compared to all other groups. While group IV patients had a statistically significant faster time to reach the maximum peak enhancement over group I and II (by 3 minutes), there was no other statistically significant difference between imaging results between groups I, II, and IV. 27 out of 128 lymphatic vessels (21%) showed dilatation, and all patients with dilated lymphatic vessels were pathologically proven to have metastases. Using the pattern of enhancement defects as the sole criterion for identifying metastatic lymph nodes during Gd-MRL interpretation, and using histopathology as the gold standard, the sensitivity and specificity were estimated to be 86% and 95%, respectively.

Conclusion

Gd-MRL can adequately depict the lymphatic system, can define sentinel lymph nodes, and has the potential to differentiate between metastatic and non-metastatic lymph nodes in breast cancer patients.     

Visualization of Intrapulmonary Lymph Vessels in Healthy and Inflamed Murine Lung Using CD90/Thy-1 as a Marker

Background

Lymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions.

Methodology/Principal Findings

Immunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located.

Conclusions/Significance

These results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions.     

The clinical importance of micrometastases within the lymphatic system in patients after total gastrectomy

Background

In spite of radical gastrectomy with resection of the lymphatic system, where no metastases are found during histopathological examination, about 30% of patients have relapse of the neoplastic process. This situation may be caused by micrometastases or isolated neoplastic cells in the lymphatic system which were not identified during a standard histopathological examination.

Aim

The aim of the study was to evaluate the clinical importance of micrometastases within the lymphatic system in patients with gastric cancer.

Materials and methods

A group of 20 patients treated for gastric cancer were subjected to retrospective analysis. Of all the patients who underwent surgery, a group with tumours classified as T1 or T2 was selected. No metastases within the lymphatic system were found in the standard evaluation – N0 mark. Paraffin-embedded blocks of lymph nodes were cut and new specimens were made, which were then stained again by means of immunohistochemistry. Antibodies against cytokeratin AE1/AE3 were used.

Results

A total of 319 lymph nodes were assessed in 20 patients in an H + E examination. After the immunohistochemical examination, micrometastases within the lymphatic system were found in 4 (20%) patients and isolated neoplastic cells in other 4 (20%) patients.

Conclusion

On the basis of numerous publications and our own material, we think that the presence of micrometastases may be related to a worse prognosis. The clinical importance of micrometastases within the lymphatic system in patients after total gastrectomy.     

Fluid Flow along Venous Adventitia in Rabbits: Is It a Potential Drainage System Complementary to Vascular Circulations?

Background

Our previous research and other studies with radiotracers showed evidence of a centripetal drainage pathway, separate from blood or lymphatic vessels, that can be visualized when a small amount of low molecular weight tracer is injected subcutaneously into a given region on skin of humans. In order to further characterize this interesting biological phenomenon, animal experiments are designed to elucidate histological and physiologic characteristics of these visualized pathways.

Methods

Multiple tracers are injected subcutaneously into an acupuncture point of KI3 to visualize centripetal pathways by magnetic resonance imaging or fluorescein photography in 85 healthy rabbits. The pathways are compared with venography and indirect lymphangiography. Fluid flow through the pathways is observed by methods of altering their hydrated state, hydrolyzing by different collagenases, and histology is elucidated by optical, fluorescein and electron microscopy.

Results

Histological and magnetic imaging examinations of these visualized pathways show they consist of perivenous loose connective tissues. As evidenced by examinations of tracers’ uptake, they appear to function as a draining pathway for free interstitial fluid. Fluorescein sodium from KI3 is found in the pathways of hind limbs and segments of the small intestines, partial pulmonary veins and results in pericardial effusion, suggesting systematical involvement of this perivenous pathway. The hydraulic conductivity of these pathways can be compromised by the collapse of their fiber-rich beds hydrolyzed by either of collagenase type I, III, IV or V.

Conclusions

The identification of pathways comprising perivenous loose connective tissues with a high hydraulic conductivity draining interstitial fluid in hind limbs of a mammal suggests a potential drainage system complementary to vascular circulations. These findings may provide new insights into a systematically distributed collagenous connective tissue with a circulatory function and their potential relevance to the nature of acupuncture meridians.