Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: Characterization of the reporter expression in lymphatic vessels of Prox1-EGFP rat. ( A ) EGFP expression patterns in adults: (i,ii) Bright light and fluorescence headshots of Prox1-EGFP mouse (left) and Prox1-EGFP rat (right) highlight the size difference between the two rodents and the strong reporter expression in their lenses. (iii–iv) Cervical and axillary lymph nodes (arrowheads), which are difficult to locate under bright light without a contrasting dye, could be easily identifiable under fluorescence in Prox1-EGFP rats. (v–vi) Fluorescent images showing lymphatic vessels in the capsular and subcapsular area of an adult lymph node. (vii–xvi) Lymphatic vessels and their luminal valves (arrowheads) are clearly visible in the bladder (vii–viii), mesentery (ix–x), and testicle (xi–xii). Schlemm’s canal (arrowhead, xiii) and limbal lymphatic vessels (arrowhead, xiv) of the same eye imaged at different focuses show strong EGFP expression. Scale bars: 500 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\rm{\mu }}$$\end{document} μ m (v–xii), 100 µm (xiii–xiv). ( B ) EGFP expression patterns in embryos: (i–iii) Compared to wild type embryo (left), Prox1-EGFP rat embryo (right) shows a strong EGFP expression in the lens, central nervous system, and liver. (iv–vi) Dermal capillary lymphatic network could be easily visualized in the back skin of embryo (E17.5). Panels (v) and (vi) are enlarged images of (iv). (vii–xxi) Bright field and fluorescence images showing lymphatic vessels in the subcutaneous area (vii–ix), thoracic cavity (x–xii), diaphragm (xiii–xv), mesentery (xvi-xviii), and bladder (xix–xxi). Scale bars: 500 µm.

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques: Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: Visualization of meningeal lymphatic vessels in Prox1-EGFP rat. Meninges from Prox1-EGFP rats were dissected and imaged as a whole mount. ( A ) Simplified diagram showing the lymphatics (green) positioned along the veins (superior sagittal and transverse sinuses) and artery (middle meningeal artery) of the rodent meninges. ( B ) Overview of whole mount meninges. SSS : Superior Sagittal Sinus, TS : Transverse Sinus, MMA : Middle Meningeal Artery, CR : Cerebellar Ring. ( C ) Lymphatic vessels were easily detected in the area of the middle meningeal artery. ( D ) Scheme of the meninges anatomy. The pia is localized on top of the brain parenchyma, the dura mater lines the skull, and the arachnoid matter lies between the pia and dura mater. The meningeal lymphatics are localized within the dura matter along the sinuses. Lymphatics were also clearly detectable in superior sagittal sinus ( E ), cerebellar ring ( F ), and transverse sinus ( G ). ( H ) Lymphatics in the cerebellar ring are enlarged in panel ( F ). Scale bars: 2 mm ( B ), 500 µm ( C,F,G ), 300 µm ( E ), 30 µm ( H ).

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques:

Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: Expression of lymphatic markers in the EGFP-positive vessels of Prox1-EGFP rat. Frozen sections from the tail ( A–C ), skin ( D–I ), and testis ( J–L ) were stained for lymphatic markers, Lyve1, Pdpn, and Prox1 to confirm the expression of lymphatic signature genes in EGFP-positive vessels. Scale bars: 100 μm ( A–I ), 50 μm ( J–L ). ( M ) Flow cytometry analyses showing a predominant Lyve1 expression in primary EGFP-positive cells (LECs) freshly isolated from lymph nodes of Prox1-EGFP rats. Cells were incubated with normal IgG control (left) or anti-Lyve1 antibody (right).

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Incubation, Control

Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: Functional analyses of the mesenteric lymphatics in Prox1-EGFP rat. ( A ) Diagram of an ex vivo study system of Prox1-EGFP lymphatic vessels. P in and P out refer to the input and output pressures, respectively, of the two cannulating pipettes. Arrow indicates the normal direction of flow. Windows for diameter tracking (by edge detection) and valve position (by densitometry) are shown by black and red boxes, respectively. Top image is a bright field image of an ex vivo vessel; bottom image is a confocal image (maximum intensity projection) of the same vessel. ( B ) Example trace showing spontaneous contractions of a lymphatic vessel when P in and P out were equal and after P out was slightly elevated. Traces were recorded under fluorescence illumination by edge detection of the outer edge of the LEC layer. The response to P out elevation is typical of wild-type vessels (see text). ( C ) Plots of changes in amplitude and frequency as functions of pressure, with both variables averaged over multiple contraction cycles. The raw amplitude and frequency data were normalized to their respective values at 10 cmH 2 O and plotted as a function of pressure. For comparison, representative data from a WT Sprague-Dawley rat mesenteric lymphatic vessel are shown as previously . ( D ) Confocal images of a passive lymphatic valve in open (top) and closed (bottom) states, as determined by the trans-valve pressure gradient. ( E ) Valve closure test conducted under fluorescence illumination with valve position determined by videodensitometry. With P in and P out equal, the valve is open; ramp-wise P out elevation with P in held constant, induces valve closure when the adverse pressure gradient exceeds 0.4 cmH 2 O. Scale bars: 100 µm.

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques: Functional Assay, Ex Vivo, Fluorescence, Comparison

Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: Visualization of host lymphatic contribution in vascularized lymph node transfer model. ( A ) Superficial inferior epigastric artery (SIEA) free flaps were isolated from EGFP − wild type Sprague-Dawley rats on a single vascular pedicle. ( B ) SIEA flap after transfer to a Prox1-EGFP recipient rat and completion of microsurgical anastomosis of the artery and vein. ( C ) Inset of the SIEA free flap using Prolene sutures, which facilitate identification upon harvest. ( D ) Gross appearance under a stereoscope of SIEA flap harvested after 30 days. Blue suture knot (arrow) marks the host-donor boundary. ( E ) EGFP signal at the inferior pole of the transferred flap at low magnification. ( F ) Enlarged image of the edge area demonstrating lymphatic vessels with EGFP + host-derived LECs. ( G–I ) Pdpn immunofluorescence assay identified host contribution to lymphangiogenesis in the donor tissue. ( J–L ) Enlarged images of boxed areas in panels ( G – I ), respectively. Scale bars: 1 mm ( D,E ), 0.5 mm ( F ), 100 µm ( G ), 50 µm ( J ).

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques: Isolation, Derivative Assay, Immunofluorescence

Journal: Scientific Reports

Article Title: Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

doi: 10.1038/s41598-017-06031-3

Figure Lengend Snippet: EGFP expression in venous valve endothelial cells of Prox1-EGFP rats. Comparative bright field ( A ) and fluorescent ( B ) images of arterial, venous, and lymphatic vessels of the abdominal wall in Prox1-EGFP adult mice. Blood-filled artery and vein alongside EGFP-positive lymphatic vessels are shown. Note that both venous valves (arrowheads) and lymphatic valves (arrows), along with lymphatic vessels, show strong EGFP expression. Similarly, bright field ( C ) and fluorescence ( D ) analyses revealed strong EGFP-positive valves (arrowheads) in the greater saphenous vein from Prox1-EGFP rats. ( E–G ) A cross-section of the greater saphenous vein showing robust EGFP signals in the closed valves ( E ). DAPI counter staining ( F ) and merged images ( G ) are also shown. ( H–J ) Fluorescent and bright field microscopic images showing EGFP-expressing opened venous valve ( H ), DAPI counter staining ( I ), and a corresponding hematoxylin-stained bright field image ( J ).

Article Snippet: The sources of the antibodies used are anti-LYVE1 antibody (AngioBio, San Diego, CA), anti-Prox1 antibody (R&D Systems, Minneapolis, MN) and anti-Pdpn antibody (PMab-2, generated by Dr. Yukinari Kato) .

Techniques: Expressing, Fluorescence, Staining