Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice

Abstract

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.

Fig. 1

Identification of lymphatic capillaries and mature lymphatic vessels. Paraffin embedded knee sections of a 6-month-old WT mouse stained with anti-podoplanin and anti-αSMA antibodies. Sections were scanned using a x 20 lens and the VS-ASW FL system. Images were observed under x 100–1000 magnification to distinguish lymphatic capillaries and mature lymphatic vessels. A) Left panel: low magnification (x 20) showing the region of interest. Areas that contain lymphatic vessels are outlined. Right panels: enlargements (x 800) of the region indicated in the left image showing a podoplanin+/αSMA-lymphatic capillary (blue arrows). B) Left panel: low magnification (x 20) showing the region of interest. Right panels: enlargements of the region indicated in the left panel showing a podoplanin+/αSMA+ mature lymphatic vessel (white arrows).

Fig. 2

Lymphatic vessels in bone. A) Left panel: low magnification micrograph (x 20) showing the region of interest. Right panel: high magnification (x 100) showing numerous lymphatic vessels in the periosteum area including lymphatic capillaries (blue arrows), mature lymphatic vessels (white arrow) and blood vessels (pink arrows). B) Left panel: low magnification (x 20) showing the region of interest. Right panel: high magnification (x 400) showing a lymphatic capillary (blue arrow) within the cortical bone of the tibia.

Fig. 3

Severe tissue damage in OA mice fed with a high fat diet. Knee joints from mice that received either MLI or sham operation, then were fed with a high fat diet or lean diet for 3 months. A) H & E stained sections show more severe OA histological changes in high fat diet fed mice, including narrowed joint space (blue arrows), clefts (red arrows), floating cartilage (green arrows), articular cartilage loss and cartilage erosion (black arrow). B) Histomorphometric analyses of H & E sections. Values are mean ± SD of 3-7 mice. * p < 0.05 vs. corresponding control mice; # p < 0.05 vs. OA mice fed a lean diet. [Q8]

Fig. 4

High fat diet reduced the area of mature lymphatic vessels in joint sections of OA mice. Knee sections from high fat diet fed mice that were described in Fig. 3. A) Sections double stained with anti-podoplanin and αSMA antibodies and scanned as described for Fig. 1. High magnification (x 100) showing decreased mature lymphatic vessels in high fat diet-OA knees (white arrows). Lymphatic capillaries and blood vessels are shown by blue and pink color arrows, respectively. B) Histomorphometric analyses were performed at the final magnification of x 100. Values are the mean ± SD of 3-7 mice. * p < 0.05 or ** p < 0.01 vs. corresponding control mice; # p < 0.05 or ## p < 0.01 vs. OA mice fed a lean diet. [Q9]

Fig. 5

VEGF-C treatment increased the area of mature lymphatic vessels in TNF-Tg RA mice. Ankle sections from 5.5-month-old TNF-Tg mice that received VEGF-C virus or Luc control virus for 4 months. Sections were double stained with anti-podoplanin and αSMA antibodies and scanned as described for Fig. 1. A) High magnification (x 100) showing increased capillary (blue arrows) and mature lymphatic vessels (white arrows) in VEGF-C-treated joints. Blood vessels are indicated by pink arrows. B) Histomorphometric analysis was performed at a final magnification of x 100. The percentage of lymphatic vessel area over inflammatory and non-inflammatory area was assessed. Values are mean + SD of 4-5 mice. * p < 0.05 vs. Luc-treated joints.

Fig. 6

Analysis of surface expression of podoplanin on joint cells from TNF-Tg mice. Cells were isolated from ankle joints of two TNF-Tg mice, stained with FITC-CD11b and PE-podoplanin antibodies and subjected to imaging cytometry analysis. A) Representative imagery from CD11b+ cells shows the surface expression of podoplanin. Yellow signal in “overlay” column is the combination of green (CD11b signal) and red (podoplanin) signals. B) Histogram shows that 66% of podoplanin+ cells express CD11b, while only 34% of podoplanin+ cells are CD11b-negative. C) Histogram overlay of podoplanin staining on CD11b+ (pink) and CD11b− (blue) cells. The mean fluorescence intensity of podoplanin CD11b+ cells is 7613, while for CD11b-cells it is 3332.