A Multiatlas Approach for Segmenting Subcortical Brain Structures using Local Patch Distance

In the diagnosis and treatment of various diseases, often segmenting the brain structures from MRI data is the key step. Since there are larger variations in the anatomical structures of the brain, segmentation becomes a crucial process. Using only the intensity information is not enough to segment structures since two or more structures may share the same tissues. Recently, the use of multiple pre-labeled images called atlases or templates are used in the process of segmentation of image data. Both single atlas and multiple atlases can be used. However, using multiple atlases in the segmentation process proves a dominant method in segmenting brain structures with challenging and overlapping structures. In this paper, we propose two multi atlas segmentation methods: Local Patch Distance Segmentation (LPDS) and Weighted Local Patch Distance Segmentation (WLPDS). These methods use local patch distance in the label fusion step. LPDS uses local patch distance to find the best patch match for label propagation. WLPDS uses local patch distance to calculate local weights. The brain MRI images from the MICCAI 2012 segmentation challenge are chosen for experimental purposes. These datasets are publicly available and can be downloaded from MIDAS. The proposed techniques are compared with existing fusion methods such as majority voting and weighted majority voting using the similarity measures such as Dice overlap (DC), Jaccard coefficient (JC) and Kappa statistics. For 20 test data sets, LPDS gives DICE=0.95±0.05, JACCARD=0.91±0.04 and KAPPA=0.94±0.07. WLPDS gives DICE=0.98±0.02, JACCARD=0.92±0.03 and KAPPA=0.95±0.04

The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses.

Lymphocyte- and leukocyte-mediated lymph node (LN) lymphatic sinus growth (lymphangiogenesis) is involved in immune responses and in diseases including cancer and arthritis. We previously discovered a 10.1.1 Ab that recognizes the lymphatic endothelial cell (LEC) surface protein mCLCA1, which is an interacting partner for LFA1 and Mac-1 that mediates lymphocyte adhesion to LECs. Here, we show that 10.1.1 Ab treatment specifically induces LEC proliferation, and influences migration and adhesion in vitro. Functional testing by injection of mice with 10.1.1 Ab but not control hamster Abs identified rapid induction of LN LEC proliferation and extensive lymphangiogenesis within 23 h. BrdU pulse-chase analysis demonstrated incorporation of proliferating LYVE-1-positive LEC into the growing medullary lymphatic sinuses. The 10.1.1 Ab-induced LN remodeling involved coordinate increases in LECs and also blood endothelial cells, fibroblastic reticular cells, and double negative stroma, as is observed during the LN response to inflammation. 10.1.1 Ab-induced lymphangiogenesis was restricted to LNs, as mCLCA1-expressing lymphatic vessels of the jejunum and dermis were unaffected by 23 h 10.1.1 Ab treatment. These findings demonstrate that 10.1.1 Ab rapidly and specifically induces proliferation and growth of LN lymphatic sinuses and stroma, suggesting a key role of mCLCA1 in coordinating LN remodeling during immune responses

10.1.1 Ab induces coordinate accumulation of lymphatic endothelial and stromal cells.

<p>Pooled LNs were enzymatically digested and stained with CD45, CD31, and Podoplanin antibodies for flow cytometry analysis. Viable cells were gated as CD45- to detect all stroma and then further gated to identify LEC (CD31+ Podoplanin+), FRC (CD31- Podoplanin+), BEC (CD31+ Podoplanin-), and DN stromal cells (CD31- Podoplanin-). A). 10.1.1 Ab-injected mice display an increased percentage of increased CD45- cells. B). Populations are shown as a percent of CD45- cells. All cell types remain proportional in the 10.1.1 Ab-injected mice indicating a coordinate expansion of LN stromal cell populations in response to 10.1.1 Ab treatment. Significance was determined using a Mann Whitney <i>U</i> test for unpaired samples. n = 6, p<0.05. Standard errors are indicated.</p

10.1.1 Ab-induced lymphangiogenesis is not due to enlargement of LECs.

<p>A). Pooled LNs were enzymatically digested and analyzed by flow cytometry. No difference in forward scatter profiles was identified between control Hamster IgG- and 10.1.1 Ab-injected mice, indicating no difference in cell size between the two treatment groups. Overlay of forward scatter profile histograms gated on viable CD45- CD31+ Podoplanin+ LECs from representative samples of Hamster IgG- and 10.1.1 Ab-injected mice are shown. B). Bar graph represents the average arithmetic mean and standard error from 2 independent experiments. n = 6.</p

10.1.1 Ab induces proliferation of LECs <i>in vitro</i>.

<p>Pooled axillary, brachial, and inguinal LNs were enzymatically digested and plated into chamber slides. Cells were treated with antibody for 5 d and stained to identify proliferating LECs. A). Immunostaining of hamster IgG-treated cultures identifies Prox1+ LECs (red) and Ki67+ proliferating cells (green), and confirms nuclear location of Prox1 and Ki67 by blue nuclear DAPI staining (right panel). B). Immunostaining of 10.1.1 Ab-treated cultures identifies a number of Prox1+ and Ki67+ proliferating LECs (e.g. arrowheads, left panel), and confirms nuclear location of Prox1 and Ki67 by DAPI staining (arrowheads, right panel). C). The Ki67+Prox1+ or Ki67+Prox1- cells from 6 preparations were counted in five fields for each sample, and the percentage of each population was determined. 10.1.1 Ab-treated samples display increased proliferating LECs compared to control Hamster IgG-treated samples. Significance was determined using a Wilcoxon Ranked Sum test for paired samples. *: p<0.05. Standard errors are indicated.</p

Lymph node lymphangiogenesis involves LEC and non-LEC proliferation.

<p>A). Schematic of the experimental timeline for the BrdU pulse-chase experiment. Mice were injected i.p. 10.1.1 Ab or control Hamster IgG at 0 h. At 16 h, mice were injected i.p. with BrdU. Two h after BrdU administration, mice were either sacrificed as part of the pulse cohort or injected i.p. with thymidine to stop further BrdU incorporation. The pulse-chase cohort was sacrificed at 38 h after Ab injection. B-E). Popliteal LN sections were stained with anti-LYVE-1 (red) and anti-BrdU (green) antibodies. White boxes on low magnification images (left panels) identify the medullary sinus regions shown at higher magnification in the right panels. LNs from Hamster IgG-injected mice from pulse (B) and pulse-chase (C) cohorts display similar LYVE-1 staining patterns and no BrdU labeling of LYVE-1+ cells. BrdU-positive LYVE-1+ LECs are identified in the pulse-labeled (D, arrowheads) and pulse chase-labeled LNs (E, arrowheads) from 10.1.1 Ab-injected mice. Most of the BrdU-positive LYVE-1- non-LEC remain clustered near the growing edge of the sinus in the pulse (D, arrows) and pulse-chase cohorts (E, arrows) indicating that these proliferating cells do not contribute directly to the sinus growth. Scale bars are indicated.</p

10.1.1 Ab or lymphocyte co-culture similarly block <i>in vitro</i> tube formation.

<p>SV-LECs were treated overnight with 30 μg/ml Hamster IgG or 10.1.1 Ab or co-cultured overnight with lymphocytes. Cells were trypsinized and plated on growth factor-reduced Matrigel at equal densities and allowed to form tubes. A). Cells were stained with Calcein AM and 4x images were analyzed. Scale bars are indicated. B). The percent area occupied by the tubes was quantified using Image J. 10.1.1 Ab treatment and lymphocyte co-culture similarly reduced tube formation potential of SV-LECs while Hamster IgG control Ab did not impact tube formation. Significance was determined using a Mann Whitney <i>U</i> test for unpaired samples. n = 5, * p<0.05.</p

10.1.1 Ab injection rapidly induces expansion of lymph node medullary sinuses.

<p>A-C). Left panels: Popliteal LN cryosections were immunostained with LYVE-1 antibody to identify lymphatic sinuses. Lymphatic sinus area was greatly increased and extended from the medulla toward the cortex in 10.1.1 Ab-injected mice (A) compared to 8.1.1 Ab (B) or Hamster IgG -injected mice (C). A-C). Right panels: Sections were stained with anti-Hamster antibody to identify binding of the 10.1.1 Ab (A), 8.1.1 Ab (B), and Hamster IgG (C). 10.1.1 Ab binds to the medullary and cortical lymphatic sinuses in 10.1.1 Ab-injected mice (A, right panel). In 8.1.1 Ab-injected samples, 8.1.1 Ab is detected binding to the lymphatic sinuses and to individual cells (B, right panel). Hamster IgG-injected samples display non-specific antibody binding of anti-hamster secondary antibody (C, right panel). Representative images are shown with the medulla located at the bottom edge of each image. Scale bars are indicated. D). Images of popliteal LNs from Hamster IgG-injected and 10.1.1 Ab-injected mice stained with LYVE-1 were quantified using NIH Image J software. 10.1.1 Ab-injected mice exhibit increased lymphatic sinus area compared to control Hamster IgG-injected mice. Six mice randomly chosen from each treatment group with three representative sections each were analyzed. Significance was determined using a Mann Whitney <i>U</i> test for unpaired samples. **p<0.005. Standard errors are indicated.</p

10.1.1 Ab induces proliferation of lymph node LECs and non-LECs.

<p>A-B). Popliteal LN sections of control Hamster IgG-injected or 10.1.1 Ab-injected mice were stained with anti-LYVE-1 antibodies (red), and anti-CD11b (green). White boxes on low magnification images (A) identify the region of medullary sinuses shown at high magnification in (B). No double-positive LYVE-1+ CD11b+ cells were identified in either treatment condition, indicating that CD11b+ cells do not directly contribute to lymphatic sinus growth. The representation or distribution of CD11b+ macrophages on each sinus (arrowheads) also did not change, indicating that sinus macrophages proportionally coat the growing lymphatic sinuses. C-D). Popliteal LN sections of control Hamster IgG-injected or 10.1.1 Ab-injected mice were stained with anti-LYVE-1 (green) and anti-Ki67 Abs (red) to identify proliferating LECs. White boxes on 10x images (C) identify the 40x regions of the medullary sinuses shown in D and E. A number of LYVE-1+ Ki67+ LECs (e.g. arrowheads) were identified near the growing edge of the medullary lymphatics in LNs from 10.1.1 Ab-injected mice but not control Hamster IgG-injected mice. Proliferating LYVE-1- Ki67+ non-LECs were clustered adjacent to the medullary lymphatic sinuses in LNs from 10.1.1 Ab-injected mice (C, arrows). E). The Ki67+ cells are DAPI-positive (e.g. compare arrowheads in D and E) confirming nuclear localization of the Ki67 immunostaining. F). Adjacent sections were stained with anti-Prox-1 (green) and anti-Ki67 (red). A number of nuclear Prox-1+ Ki67+ proliferating LECs (e.g. arrowheads) were identified in the same regions of the growing lymphatic sinuses in LNs from 10.1.1 Ab-injected but not hamster IgG-injected mice. Scale bars are indicated.</p