The human spleen is equipped with an organ-specific microcirculation. The initial part of the venous circulation is formed by spleen-specific large microvessels, the sinuses. Sinuses eventually fuse to form venules and veins. For more than 170 years there have been debates, whether splenic red pulp capillaries join sinuses, i.e., whether the microcirculation is closed or open—or even simultaneously closed and open. We have now solved this question by three-dimensional reconstruction of a limited number of immunostained serial sections of red and white pulp areas, which were visualized in virtual reality. Splenic capillaries have special end structures exhibiting multiple small diverging endothelial cell processes, which always keep a certain distance to the walls of sinuses. Only very few capillary ends were difficult to diagnose. Positive identification of these end structures permits to conclude that the human splenic microcirculation is entirely open. This is also true for the perifollicular capillary network and for capillaries close to red pulp venules. Follicles are supplied by a relatively dense open perifollicular capillary net, which is primarily, but not exclusively, fed by sheathed and few non-sheathed capillaries from the surrounding red pulp network.

3D reconstruction is a challenging current topic in medical research. We perform 3D reconstructions from serial sections stained by immunohistological methods. This paper presents an immersive visualization solution to quality control (QC), inspect, and analyze such reconstructions. QC is essential to establish correct digital processing methodologies. Visual analytics, such as annotation placement, mesh painting, and classification utility, facilitates medical research insights. We propose a visualization in virtual reality (VR) for these purposes. In this manner, we advance the microanatomical research of human bone marrow and spleen. Both 3D reconstructions and original data are available in VR. Data inspection is streamlined by subtle implementation details and general immersion in VR.

AbstractThe spleen is a secondary lymphoid organ present in all vertebrates, which monitors the blood. It consists of sessile stroma cells (fibroblasts), migratory cells of the immune system, blood‐filled spaces and unique vessels. Splenic functions and microanatomy differ according to the species investigated. Certain functions such as immunological monitoring of blood‐borne antigens, destruction of intravasal microorganisms and aged or abnormal blood cells, are more or less invariant. In rodents and humans, the spleen is composed of two large compartments, the white pulp and the red pulp. The white pulp harbours dense accumulations of more or less migratory lymphocytes and antigen‐presenting cells, which crawl on a meshwork of specialised fibroblasts. The red pulp is composed of connective tissue containing arterioles, sheathed and postsheath capillaries, sinuses and venules/veins. The sinuses represent a spleen‐specific initial part of the venous circulation. The connective tissue of the red pulp cords is filled with blood and represents the only place where an open circulation occurs in the body.In addition to describing the microanatomical compartments of rat and human spleens, the article deals with the course of acute B lymphocyte immune reactions in rat and mouse spleens and with the consequences of splenectomy in humans.Key ConceptsThe spleen monitors the antigen composition of the blood.The monitoring is predominantly carried out by lymphocytes recirculating through the spleen in the white pulp and by sessile macrophages in the red pulp cords.Specialised fibroblasts recruit lymphocytes and macrophages to their compartments with the help of several mechanisms, such as secretion of chemokines or expression of certain adhesion molecules.The splenic red pulp cords are the only location in the body with an open circulation. This means that blood flows in spaces not lined by any barrier‐forming cells such as endothelia.The open circulation thus permits a direct contact between the blood and resident macrophages.In rodents and humans, the spleen contains a special type of B lymphocytes, termed 'B cells of marginal zone (MZ) phenotype'. These cells represent preactivated B cells, which fulfil memory functions and/or recognise polysaccharide antigens. They are easily activated to become antibody‐secreting plasma cells.In humans, splenectomy leads to the loss of this B cell type in the blood and to a reduced resistance against bacteria with polysaccharide capsules. In addition, the most effective phagocyte compartment of the body, the splenic red pulp, is lost. This necessitates special precautions, especially in children, to avoid overwhelming postsplenectomy infections.

Plasma cells (PCs) in human palatine tonsils are predominantly located in the germinal centres (GCs), in the subepithelial space and near the deep connective tissue septa surrounding each crypt. We analysed the location, phenotype, and proliferation of GC PCs by immunohistology comparing them to PCs in the other two locations. Most PCs in GCs were strongly positive for CD38, CD138, CD27, IRF4, and intracellular (ic) IgG. They often accumulated in the basal light zone, but could also be found scattered in the entire light zone. In addition, rows of PCs occurred at the surface of the GC bordering the mantle zone, i.e., in the outer zone, and at the surface of the dark zone. The latter cells were often continuous with PCs in the extrafollicular area. The vast majority of GC PCs were negative for Ki-67. Only a few Ki-67 + plasmablasts, predominantly icIgG + or icIgM + , were found inside GCs. In certain GCs PCs accumulated around capillaries and the adjacent perikarya of follicular dendritic cells (FDCs). Newly formed PCs might migrate from the basal to the superficial part of the light zone and then back to the dark zone surface to leave the GC. This guarantees an even distribution of secreted Ig for exchange with immune complexes on FDCs. The surface of the dark zone may also be an exit site for Ki-67 + CD30 + B lymphoblasts, which seed perifollicular and extrafollicular sites. We speculate that these cells tend to downmodulate CD20 and activation-induced deaminase and further up-regulate CD30 when developing into pre-plasmablasts.

We reconstructed serial sections of a representative adult human spleen to clarify the unknown arrangement of the splenic microvasculature, such as terminal arterioles, sheathed capillaries, the red pulp capillary network and venules. The resulting 3D model was evaluated in virtual reality (VR). Capillary sheaths often occurred after the second or third branching of a terminal arteriole and covered its capillary side or end branches. The sheaths started directly after the final smooth muscle cells of the arteriole and consisted of cuboidal CD271 ++ stromal sheath cells surrounded and infiltrated by B lymphocytes and macrophages. Some sheaths covered up to four sequential capillary bifurcations thus forming bizarre elongated structures. Each sheath had a unique form. Apart from symmetric dichotomous branchings inside the sheath, sheathed capillaries also gave off side branches, which crossed the sheath and freely ended at its surface. These side branches are likely to distribute materials from the incoming blood to sheath-associated B lymphocytes and macrophages and thus represent the first location for recognition of blood-borne antigens in the spleen. A few non-sheathed bypasses from terminal arterioles to the red pulp capillary network also exist. Red pulp venules are primarily supplied by sinuses, but they also exhibit a few connections to the capillary network. Thus, the human splenic red pulp harbors a primarily open microcirculation with a very minor closed part.

Stromal capillary sheath cells in human spleens strongly express CD271, the low affinity nerve growth factor receptor p75. Serial sections of a representative adult human spleen were double-stained for CD271 versus smooth muscle alpha actin (SMA) plus CD34 to visualise capillary sheaths, the arterial tree and endothelial cells by transmitted light. Preliminary three-dimensional (3D) reconstructions of single regions were inspected in virtual reality (VR). This method showed that a large number of CD271+ sheaths occur in a post-arteriolar position often surrounding capillaries located close to divisions of arterioles. The length and diameter of capillary sheaths are rather heterogeneous. Long sheaths were observed to accompany one or two generations of capillary branches. We hypothesise that human splenic capillary sheaths may attract recirculating B-lymphocytes from the open circulation of the red pulp to start their migration into white pulp follicles along branches of the arterial tree. In addition, they may provide sites of interaction among sheath macrophages and B-lymphocytes. Our innovative approach allows stringent quality control by inserting the original immunostained serial sections into the 3D model for viewing and annotation in VR. Longer series of sections will allow to unequivocally localise most of the capillary sheaths in a given volume.

We have reconstructed small parts of capillary networks in the human splenic white pulp using serial sections immunostained for CD34 alone or for CD34 and CD271. The three-dimensional (3D) models show three types of interconnected networks: a network with very few long capillaries inside the white pulp originating from central arteries, a denser network surrounding follicles plus periarterial T-cell regions and a network in the red pulp. Capillaries of the perifollicular network and the red pulp network have open ends. Perifollicular capillaries form an arrangement similar to a basketball net located in the outer marginal zone. The marginal zone is defined by MAdCAM-1+ marginal reticular stromal cells. Perifollicular capillaries are connected to red pulp capillaries surrounded by CD271+ stromal capillary sheath cells. The scarcity of capillaries inside the splenic white pulp is astonishing, as non-polarised germinal centres with proliferating B-cells occur in adult human spleens. We suggest that specialized stromal marginal reticular cells form a barrier inside the splenic marginal zone, which together with the scarcity of capillaries guarantees the maintenance of gradients necessary for positioning of migratory B- and T-lymphocytes in the human splenic white pulp.

Histological serial sections allow for 3D representation of anatomical structures in microscopic to mesoscopic range. However, due to the nature of the acquisition, they suffer from severe anisotropy: 14-to-1 in a single average microscopic paraffin section. We present an interpolation method based on optical flow and show that standard interpolation methods are less suited for serial sections. With our non-linear interpolation approach we are able to represent the "movement" of image parts that are of interest. This allows for better 3D reconstructions and further insights in microanatomy.

The form and exact function of the blood vessel network in some human organs, like spleen and bone marrow, are still open research questions in medicine. In this paper, we propose a method to register the immunohistological stainings of serial sections of spleen and bone marrow specimens to enable the visualization and visual inspection of blood vessels. As these vary much in caliber, from mesoscopic (millimeter-range) to microscopic (few micrometers, comparable to a single erythrocyte), we need to utilize a multi-resolution approach.Our method is fully automatic; it is based on feature detection and sparse matching. We utilize a rigid alignment and then a non-rigid deformation, iteratively dealing with increasingly smaller features. Our tool pipeline can already deal with series of complete scans at extremely high resolution, up to 620 megapixels. The improvement presented increases the range of represented details up to smallest capillaries. This paper provides details on the multi-resolution non-rigid registration approach we use. Our application is novel in the way the alignment and subsequent deformations are computed (using features, i.e. "sparse"). The deformations are based on all images in the stack ("global").We also present volume renderings and a 3D reconstruction of the vascular network in human spleen and bone marrow on a level not possible before. Our registration makes easy tracking of even smallest blood vessels possible, thus granting experts a better comprehension.A quantitative evaluation of our method and related state of the art approaches with seven different quality measures shows the efficiency of our method. We also provide z-profiles and enlarged volume renderings from three different registrations for visual inspection. (C) 2016 Elsevier B.V. All rights reserved.

The arrangement of microvessels in human bone marrow is so far unknown. We combined monoclonal antibodies against CD34 and against CD141 to visualise all microvessel endothelia in 21 serial sections of about 1 cm2 size derived from a human iliac crest. The specimen was not decalcified and embedded in Technovit® 9100. In different regions of interest, the microvasculature was reconstructed in three dimensions using automatic methods. The three-dimensional models were subject to a rigid semiautomatic and manual quality control. In iliac crest bone marrow, the adipose tissue harbours irregularly distributed haematopoietic areas. These are fed by networks of large sinuses, which are loosely connected to networks of small capillaries prevailing in areas of pure adipose tissue. Our findings are compatible with the hypothesis that capillaries and sinuses in human iliac crest bone marrow are partially arranged in parallel.

The microanatomical structure of the spleen has been primarily described in mice and rats. This leads to terminological problems with respect to humans and their species-specific splenic microstructure. In mice, rats and humans the spleen consists of the white pulp embedded in the red pulp. In the white pulp, T and B lymphocytes form accumulations, the periarteriolar lymphatic sheaths and the follicles, located around intermediate-sized arterial vessels, the central arteries. The red pulp is a reticular connective tissue containing all types of blood cells. The spleen of mice and rats exhibits an additional well-delineated B-cell compartment, the marginal zone, between white and red pulp. This area is, however, absent in human spleen. Human splenic secondary follicles comprise three zones: a germinal centre, a mantle zone and a superficial zone. In humans, arterioles and sheathed capillaries in the red pulp are surrounded by lymphocytes, especially by B cells. Human sheathed capillaries are related to the splenic ellipsoids of most other vertebrates. Such vessels are lacking in rats or mice, which form an evolutionary exception. Capillary sheaths are composed of endothelial cells, pericytes, special stromal sheath cells, macrophages and B lymphocytes. Human spleens most probably host a totally open circulation system, as connections from capillaries to sinuses were not found in the red pulp. Three stromal cell types of different phenotype and location occur in the human white pulp. Splenic white and red pulp structure is reviewed in rats, mice and humans to encourage further investigations on lymphocyte recirculation through the spleen.

The spleen is one of the organs, where the micro-structure and the function on that level are not completely understood. It was for example only recently found that is has an open circulation, which distinguishes it from all other organs. Imaging the complete vascular network from the arteries to open-ended capillaries would greatly facilitate research in this area. The structure of such tissue is best uncovered using immunehistological staining. This can however only be applied to thin tissue sections and larger structures span several slices. Due to the deformation induced when cutting the specimen, standard registration algorithms cannot be used to merge the images into a volume. We propose a specialized matching algorithm to robustly determine corresponding regions in the images. After a rigid alignment of the scans, we use a cubic B-spline to deform and align the images. During this process we minimize the total deformation to produce as accurate results as possible.

At least three phenotypically and morphologically distinguishable types of branched stromal cells are revealed in the human splenic white pulp by subtractive immunohistological double-staining. CD271 is expressed in fibroblastic reticulum cells of T-cell zones and in follicular dendritic cells of follicles. In addition, there is a third CD2711- and CD271+/) stromal cell population surrounding T-cell zones and follicles. At the surface of follicles the third population consists of individually variable partially overlapping shells of stromal cells exhibiting CD90 (Thy-1), MAdCAM-1, CD105 (endoglin), CD141 (thrombomodulin) and smooth muscle α-actin (SMA) with expression of CD90 characterizing the broadest shell and SMA the smallest. In addition, CXCL12, CXCL13 and CCL21 are also present in third-population stromal cells and/or along fibres. Not only CD27+ and switched B lymphocytes, but also scattered IgD++ B lymphocytes and variable numbers of CD4+ T lymphocytes often occur close to the third stromal cell population or one of its subpopulations at the surface of the follicles. In contrast to human lymph nodes, neither podoplanin nor RANKL (CD254) were detected in adult human splenic white pulp stromal cells. The superficial stromal cells of the human splenic white pulp belong to a widespread cell type, which is also found at the surface of red pulp arterioles surrounded by a mixed T-cell/B-cell population. Superficial white pulp stromal cells differ from fibroblastic reticulum cells and follicular dendritic cells not only in humans, but apparently also in mice and perhaps in rats. However, the phenotype of white pulp stromal cells is species-specific and more heterogeneous than described so far.

The microvasculature of human spleens is still incompletely understood. Two enigmatic types of red pulp microvessels, penicillar arterioles and sheathed capillaries, have already been described in the nineteenth century without gaining much attention afterwards. We performed a detailed analysis of sheathed capillaries to clarify the cellular composition of their sheaths by immunohistological double-staining experiments. Capillary sheaths comprise three different cell types, namely specialized cuboidal CD271++ inner sheath cells surrounded by CD271− macrophages and accumulations of B lymphocytes. The CD271++ inner sheath cells express the chemokine CXCL13 in a unique single dot pattern. Sheath-associated B lymphocytes consist of IgM+, IgD++, and of “switched” cells. T lymphocytes do not accumulate in pericapillary sheaths. The predominant sheath-associated macrophage population is CD163−CD68+ and thus differs from the majority of red pulp macrophages. The sheath-associated macrophages strongly express CD169 only in perifollicular sheaths, but not in sheaths located deeper in the red pulp. IgM+, IgD++, and “switched” B cells are also closely associated with red pulp arterioles characterized by the expression of smooth muscle actin in muscle cells and in branched periarteriolar stromal cells. Capillary sheaths are observed in a post-arteriolar position and appear to be of limited length. We suggest to change the term “Vagina periarteriolaris makrophagocytica” of the international histological and embryological terminologies to “Vagina pericapillaris.”

A technique for embedding human undecalcified tooth specimens in Technovit® 9100 was developed, which permits immunohistological evaluation of pulp tissue in serial ground sections. Human molars were divided into 14-18 sections of about 23 µm thickness. Immunohistological double staining for S-100 and CD34 revealed unique associations of myelinated nerve fibre bundles with arterioles, which continued through the entire tooth pulp. These arterioles were not only accompanied by, but partially or totally enveloped in longitudinally orientated myelinated nerve fibre bundles. We speculate that this unique arrangement may mechanically support the arterioles and alleviate detection or regulation of their contraction state by sensory nerve cells.

AbstractThe spleen is a secondary lymphoid organ present in all vertebrates, which monitors the blood. It consists of sessile stroma cells (fibroblasts), migratory cells of the immune system and blood‐filled spaces and vessels. Splenic functions and microanatomy differ according to the species investigated. Certain functions such as immunological monitoring of bloodborne antigens, destruction of intravasal microorganisms and aged or abnormal blood cells, are more or less invariant. In rodents and humans the spleen is composed of two large compartments, the white pulp and the red pulp. The white pulp harbours dense accumulations of more or less migratory lymphocytes and antigen‐presenting cells, which crawl on a meshwork of specialised fibroblasts. The red pulp is composed of connective tissue cords containing arterioles and capillaries and of sinuses, which represent a spleen‐specific initial part of the venous circulation. The connective tissue of the red pulp cords is filled with blood and represents the only place where an open circulation occurs in the body. In addition, the cords harbour a dense population of specialised macrophages and some plasma cells. The article describes the microanatomical compartments of rat and human spleens, the course of B lymphocyte immune reactions in rodent spleens and the consequences of splenectomy in humans.Key Concepts:The spleen monitors the antigen composition of the blood.The monitoring is predominantly carried out by lymphocytes recirculating through the spleen in the white pulp and by sessile macrophages in the red pulp cords.Specialised fibroblasts recruit lymphocytes and macrophages to their compartments with the help of several mechanisms, such as secretion of chemokines or expression of certain adhesion molecules.The splenic red pulp cords are the only location in the body with an open circulation. This means that blood flows in spaces not lined by any barrier‐forming cells such as endothelia.The open circulation thus permits a direct contact between the blood and resident macrophages.In rodents and humans the spleen contains a special type of B lymphocytes, the marginal zone cells. These cells recognise polysaccharide antigens and are easily activated to become antibody‐secreting plasma cells.In humans, splenectomy leads to the loss of marginal zone‐type B cells in the blood and to a reduced resistance against bacteria with polysaccharide capsules. This is especially relevant in children.The susceptibility to overwhelming post‐splenectomy infections in humans may be due to loss of the marginal zone and its special stromal cells in combination with loss of the large phagocyte compartment in the splenic red pulp.

The structure of germinal centres (GCs) in human secondary lymphatic organs has not been thoroughly investigated until now. We stained follicular dendritic cells (FDCs) in serial sections of human hyperplastic tonsils and spleens to compare the morphology of GCs in fulminant immune reactions and quiescence. Detection of CD35, CD21, CD23 and the target of mAb CNA.42 confirmed that full-blown human tonsil GCs may consist of four regions, the dark zone, the basal and apical light zone and the outer zone. The outer zone was, however, not a constant feature of tonsillar GCs and existed only in a minority of follicles in most specimens. Thus, between 3 and 60% of tonsil GCs with a CD23(+) apical light zone exhibited an outer zone in individual specimens. FDCs in tonsil GCs appeared to be extremely sensitive to mechanical stress during surgery. In contrast to tonsils, seven of the eight adult spleens did not exhibit asymmetric polarized GCs, but only symmetric GCs without dark and light zones or follicles with few GC B cells. Some specimens apparently only contained primary follicles after conventional staining, but on closer inspection a homogenous hyaline extracellular material deposited among the FDCs indicated that a GC had been present. Our study demonstrates that the structure of GCs varies in different human secondary lymphatic organs most likely depending on the local antigenic challenge.

It has long been debated whether the red pulp of human spleens harbors an open or a closed microcirculation or both. To solve this issue, the authors differentially stained the endothelium in red pulp arterial microvessels and in venous sinuses using brightfield and fluorescence immunohistology with reagents against CD34 and CD141. Three-dimensional models of red pulp arterial microvessels and sinuses were derived from serial double-stained paraffin sections with the help of license-free open-access software. In each model, arterial microvascular ends were traced and verified by reference to the original serial sections. In total, 142 ends were analyzed in the specimens of three individuals. None of these ends was connected to a sinus, suggesting that the human splenic red pulp harbors an entirely open circulatory system. Thus, the spleen is the only human organ where blood passes through spaces not lined by endothelia or other barrier-forming cells.

Background CD1d is a nonpolymorphic MHC class I-like molecule which presents nonpeptide ligands, e.g. glycolipids, to NKT cells. These cells are known to have multiple effects on innate and adaptive immune responses and on the development of pathological conditions. In order to analyze CD1d expression and function in the rat, the first rat CD1d-specific monoclonal antibodies (mAbs) were generated. Methodology/Principal Findings Two mAbs, WTH-1 and WTH-2, were generated which bound equally well to cell surface-expressed rat and mouse CD1d. Their non-overlapping epitopes were mapped to the CD1d heavy chain. Flow cytometry and immunohistological analyses revealed a nearly identical degree and pattern of CD1d expression for hematopoieitic cells of both species. Notable is also the detection of CD1d protein in mouse and rat Paneth cells as well as the extremely high CD1d expression in acinar exocrine cells of the rat pancreas and the expression of CD4 on rat marginal zone B cells. Both mAbs blocked α-galactosylceramide recognition by primary rat and mouse NKT cells. Interestingly, the two mAbs differed in their impact on the activation of various autoreactive T cell hybridomas, including the XV19.2 hybridoma whose activation was enhanced by the WTH-1 mAb. Conclusions/Significance The two novel monoclonal antibodies described in this study, allowed the analysis of CD1d expression and CD1d-restricted T cell responses in the rat for the first time. Moreover, they provided new insights into mechanisms of CD1d-restricted antigen recognition. While CD1d expression by hematopoietic cells of mice and rats was extremely similar, CD1d protein was detected at not yet described sites of non-lymphatic tissues such as the rat exocrine pancreas and Paneth cells. The latter is of special relevance given the recently reported defects of Paneth cells in CD1d−/− mice, which resulted in an altered composition of the gut flora.