Delineation of the healthy rabbit duodenum by immunohistochemistry - A short communication.

The duodenum acts as a vital organ that performs fundamental physiological functions like digestion and nutrient absorption. Situated in the lower abdomen, the duodenum is located between the stomach and the jejunum. Usually, the duodenum is divided into four anatomical portions. We here compare paraffin embedded and cryosections of the healthy rabbit duodenum for research purposes. This analysis evaluates the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting extracellular matrix markers collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and proliferation marker ki67 as well as inflammatory marker PAR-2. Subsequent recommendations are provided based on our findings. Furthermore, the advantage of an antigen retrieval step in immunohistochemical labelling of paraffin sections was demonstrated and confirmed with an isotype negative control. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labellings was performed in serial sections, showing that adjacent to the circular muscle of the duodenum, the connective tissue was composed of collagen I and fibronectin, while the artery and vein walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where particularly a type of gland adjacent to Brunner's glands showed prominent PAR-2 positive areas, while the Brunner's glands themselves were PAR-2 negative. Proliferating ki67 positive cells facing the lumen were highly abundant in all kinds of glands except for the Brunner's glands. This effort serves to furnish the research community with reference imagery pertinent to scientists opting for the rabbit duodenum model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rat intestinal tracts, or even offer insights into the human context.

Delineation of the healthy rabbit tongue by immunohistochemistry - A technical note.

In the oral cavity the tongue is an important muscular organ that supports the swallowing of food and liquids. It is responsible for the sense of taste, based on the many different taste buds it contains. Research in the field of tongue diseases demands for suitable preclinical models. The healthy rabbit tongue may therefore serve as baseline and reference for the pathological situation. With this consideration, we covered the fixation and histological stainings as well as the immunohistochemical labelling of the healthy rabbit tongue. In this technical note, initial choice of the fixative is discussed, with a comparison of formalin fixation and subsequent paraffin embedding versus cryopreservation. Moreover, we delineate the effect of an antigen retrieval step for formalin fixation by several examples. Finally, we provide ECM markers collagen I, collagen III, fibronectin, α-SMA and elastin staining as well as ki67 for proliferative status and PAR-2 protein expression as a marker for inflammatory status and nociception in tongue sections, mainly from the tongue body. Technically, we found superiority of paraffin sections for collagen I, collagen III, fibronectin, ki67 and α-SMA labelling, for selected detections systems. As for ECM components, the lamina propria was very rich in collagen and fibronectin, while the muscular body of the tongue showed only collagen and fibronectin positive areas between the muscle fibers. Moreover, α-SMA was clearly expressed in the walls of arteries and veins. The inflammatory marker PAR-2 on the other hand was prominently expressed in the salivary glands and to some extent in the walls of the vessels. Particular PAR-2 expression was found in the excretory ducts of the tongue. This technical note has the aim to provide baseline images that can be used to compare the pathological state of the diseased rabbit tongue as well as for inter-species comparison, such as mouse or rat tongue. Finally, it can be used for the comparison with the human situation.

Delineation of the healthy rabbit tonsil by immunohistochemistry - A short communication.

Situated in the oral cavity, the rabbit palatine tonsils are part of the mucosal immune system and help to defend the body against foreign pathogens. Expressed as two oval protrusions in the wall of the oropharynx, the rabbit palatine tonsils are characterized by excretory ducts and trabeculae. We here compare paraffin embedded and cryosections of the healthy rabbit tonsils. This analysis centers on evaluating the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and ki67. Subsequent recommendations are provided based on our findings. Furthermore, we demonstrate the advantage of an antigen retrieval step in immunohistochemical labeling of paraffin sections. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labelings was furthermore performed in serial sections, showing that adjacent to the excretory ducts, the tonsillar tissue was particularly composed of collagen I and fibronectin, while the vessel walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where a small fraction of the cells found in the tonsillar connective tissue were PAR-2 positive (probably a subpopulation of mast cells), as well as the lumen of some excretory ducts and trabeculae. Collagen III on the other hand was only weakly expressed in the tonsils. Proliferating ki67 positive cells were rare. This endeavor serves to furnish the scientific community with reference imagery pertinent to researchers opting for the rabbit palatine tonsil model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rodent tonsils, or even offer insights into the human context.

Delineation of the healthy rabbit heart by immunohistochemistry - A technical note.

Heart failure poses a big health problem and may result from obesity, smoking, alcohol and/or growing age. Studying pathological heart tissue demands accurate histological and immunohistochemical stainings in animal models, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy heart stainings and labeling are required, in order to provide a reference for the pathological situation. Heart and brain tissue of a healthy rabbit were collected, and different histological key steps were compared, such as paraffin embedding after formalin fixation versus cryopreservation; an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or a chromogenic with a fluorescent detection system, respectively. Using serial sections, we stained the same morphological structure with classic approaches (HE, Masson Goldner Trichrome (GT) and Elastica van Gieson (EL)) and with different markers, including collagen I, collagen III, fibronectin, α-SMA, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, and ki67 for proliferating cells. Differences between conditions were quantitatively assessed by measuring the color intensity. Generally, cryosections exhibited a more prominent signal intensity in immunohistochemically labeled sections than in paraffin sections, but the strong staining was slurry, which sometimes impeded proper identification of morphological structures, particularly at higher magnifications. In addition, the advantage of an AR step was observed when compared to the condition without AR, where signal intensities were significantly lower. Different stainings of the heart arteries and the myocardium revealed a clear distribution of extracellular matrix components, with prominent collagen III in the artery wall, but an absence of collagen III in the myocardium. Moreover, paraffin-embedded sections provided more distinct structures compared to cryosections after collagen III, ki67, fibronectin, and α-SMA labeling. As for the Purkinje cells that were depicted in the heart and the cerebellum (Purkinje neurons), we found GT staining most suitable to depict them in the heart, while HE as well as EL staining was ideal to depict Purkinje neurons in the cerebellum. In sum, we provide useful reference images with different stainings for researchers using the rabbit heart or brain model. Such images can help to decide which of the immunohistochemical protocols are valuable to reach a specific aim. Recommendations are given for the best visualization of the target structures and specific (immunohistochemical) staining.

High-Affinity Cu(I)-Chelator with Potential Anti-Tumorigenic Action-A Proof-of-Principle Experimental Study of Human H460 Tumors in the CAM Assay.

Human lung cancer ranks among the most frequently treated cancers worldwide. As copper appears critical to angiogenesis and tumor growth, selective removal of copper represents a promising strategy to restrict tumor growth. To this end, we explored the activity of the novel high-affinity membrane-permeant Cu(I) chelator PSP-2 featuring a low-zeptomolar dissociation constant. Using H460 human lung cancer cells, we generated small tumors on the chorioallantoic membrane of the chicken embryo (CAM assay) and studied the effects of topical PSP-2 application on their weight and vessel density after one week. We observed a significant angiosuppression along with a marked decrease in tumor weight under PSP-2 application compared to controls. Moreover, PSP-2 exposure resulted in lower ki67+ cell numbers at a low dose but increased cell count under a high dose. Moreover, HIF-1α+ cells were significantly reduced with low-dose PSP-2 exposure compared to high-dose and control. The total copper content was considerably lower in PSP-2 treated tumors, although statistically not significant. Altogether, PSP-2 shows promising potential as an anti-cancer drug. Nevertheless, further animal experiments and application to different tumor types are mandatory to support these initial findings, paving the way toward clinical trials.

Probing Vasoreactivity and Hypoxic Phenotype in Different Tumor Grafts Grown on the Chorioallantoic Membrane of the Chicken Embryo In Ovo Using MRI.

Tumor grafts grown on the chorioallantoic membrane (CAM) of chicken embryos represent a transition between cell culture and mammalian in vivo models. Magnetic resonance imaging (MRI) started to harness this potential. Functional gas challenge is feasible on the CAM. Using quantitative T1 and T2* mapping, we characterized the response of MC-38 colon, A549, and H460 adeno-carcinoma cell grafts to hypercapnic (HC) and hypercapnic-hyperoxic (HCHO) gas challenges, pertaining to the grafts' vascular and oxygenation phenotypes. MR imaging revealed that larger T1 and T2* were located in the center of H460 and MC-38 tumors. Quantitative analysis showed a significant reduction in T1 and a significant increase in T2* in response to HCHO for A549 grafts, while H460 and MC-38 tumors did not respond to either gas challenge. Different tumor grafts respond differentially to HC and HCHO conditions. A549 tumor grafts, with higher vessel density and smaller tumor diameter compared with H460 and MC-38 grafts, had a significant response in T1 for HCHO and T2* increased slightly during HC and significantly under HCHO, consistent with a normoxic phenotype and functional vasoreactivity. Therefore, gas challenges enable differential characterization of tumor grafts with respect to their vascular and oxygenation status.

Electrospun tube reduces adhesion in rabbit Achilles tendon 12 weeks post-surgery without PAR-2 overexpression.

One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (- 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury.

Delineation of the healthy rabbit liver by immunohistochemistry - A technical note.

Liver diseases pose a big global health problem and liver failure may result from viral infection, overnutrition or tumors. Studying pathologic liver tissue demands for accurate and specific histological stainings and immunohistochemical labellings, including chromogenic and fluorescent approaches. Moreover, a reliable set of healthy liver stainings and labellings are required, to provide a baseline or reference for the pathological situation. Here, we used the liver tissue of a healthy rabbit and compared different histological key steps, such as paraffin embedding after formalin fixation versus cryopreservation; or an antigen retrieval (AR) step in processing paraffin sections versus the same procedure without AR; or chromogenic with fluorescent detection system, respectively. Moreover, we provide images of serial sections, where we stained the same morphological structure with different markers, including collagen I, collagen III, fibronectin, α-SMA, elastin, protease-activated receptor-2 (PAR-2) which is an inflammation-related marker, ki67 for proliferating cells, and orcein (as negative control for pathological aberrations like Wilson disease). Differences between conditions were quantitatively assessed by measuring the colour intensity. Generally, we observed that cryosections exhibited a stronger signal intensity in immunohistochemically labelled sections than in paraffin sections; however, the strong staining got slurred, which sometimes hampered proper identification of morphological structures at higher magnifications. Moreover, there was a clear increase in signal intensity for paraffin sections when an AR step was performed compared to condition without AR. Results for mouse isotype staining as a negative control clearly supported those findings. Different stainings of the portal triad, the central vein and the bile ducts revealed a clear-cut distribution of extracellular matrix components, with prominent fibronectin and elastin around the lumen of the central vein as well as a patchy PAR-2 expression. As for the bile ducts, complete absence of α-SMA and PAR-2 was found at the margins, however, collagen I expression and elastin were positive and showed a strong signal. Like this, we provide useful and valuable reference images for researchers using the rabbit liver model. It may help to decide which of the immunohistochemical protocols are valuable to reach a certain aim and which protocols lead to the best visualization of the target structure.

Delineation of the healthy rabbit kidney by immunohistochemistry - A technical note.

Pre-clinical animal models are needed to investigate and study kidney injuries and diseases. The rabbit kidney model is frequently used because various important parameters can be assessed with it. For example, histology and immunohistochemistry are indispensable as tissue morphology and composition can be investigated qualitatively as well as quantitatively. Here, different histological and immunohistochemical stainings were performed in the rabbit healthy naïve kidney tissue. First, overnight formalin fixation followed by paraffin embedding and cryopreservation with a subsequent 10-minute formalin fixation prior to staining were compared. Cryosections showed a more pronounced staining pattern, with clear borders at low magnifications, but blurred borders at higher magnifications. Then, antigen retrieval (AR) for paraffin embedded sections resulted in more prominent corresponding signals compared to stainings without AR. Moreover, several advantages and disadvantages of chromogenic versus immunofluorescence stainings were considered. Chromogenic staining was advantageous compared to immunofluorescence for collagen I and III, and to a minor degree for fibronectin. Finally, distinct structures, such as the pelvis, the calices, the glomeruli and tubuli, were stained in serial sections with diverse immunohistochemical stainings in order to delineate their composition. The following stainings were performed: standard Haematoxylin&Eosin and Elastica van Gieson staining, collagen I, collagen III, fibronectin, α-SMA, ki-67 and protease-activated receptor-2 (PAR-2). While chromogenic stainings of collagen I and collagen III were particularly useful to depict kidney structures in paraffin sections compared with cryosections, cryosections immunofluorescently stained for α-SMA were superior to paraffin sections, particularly at higher magnifications. With regard to specific structures, we found renal vessel walls positive for fibronectin and α-SMA, while the Bowman's capsule was only positive for fibronectin and α-SMA showed only tiny spots. The mesangial cells of the glomeruli and the distal tubuli were PAR-2 positive, while the proximal tubuli were PAR-2 negative.

Delineation of the healthy rabbit lung by immunohistochemistry - a technical note.

Investigation and studies of pulmonary diseases and injuries require pre-clinical animal models. The rabbit lung model is widely used and allows for a diverse set of readouts. Among them, histology and immunohistochemistry are of invaluable merit because qualitative and quantitative information about tissue morphology and composition can be easily obtained. In this technical note, we performed several histological and immunohistochemical stainings in the rabbit healthy naïve lung tissue. Overnight formalin fixation with subsequent paraffin embedding was compared to cryopreservation with a subsequent 10-minute formalin fixation prior to staining. Antigen retrieval (AR) for paraffin embedded sections proved to enhance the corresponding signals compared to analogous staining without AR. Advantages and disadvantages of chromogenic versus immunofluorescence stainings were discussed. In addition, several morphological structures, such as the intrapulmonary bronchus with its mucosal folds, the pulmonary artery, the alveoli and the lymph nodes, were stained with various stainings at the same site in order to give a comprehensive picture of their composition. Besides Haematoxylin&Eosin and Elastica van Gieson staining, collagen I, collagen III, fibronectin, α-SMA, ki-67 and protease-activated receptor-2 (PAR-2) immunohistochemistry was performed. Collagen I, collagen III and fibronectin expression was positive at the outer rim of the pulmonary arteries, while the inner rim was collagen III positive. Moreover, the fibronectin staining in the intrapulmonary bronchus showed an opposite trend when compared to the collagen III staining. The alveoli exhibited PAR-2 expression, while PAR-2 was not expressed in lymph nodes of the healthy rabbit lung.

Tumor grafts grown on the chicken chorioallantoic membrane are distinctively characterized by MRI under functional gas challenge.

Recently, a tumor model based on the chorioallantoic membrane (CAM) was characterized structurally with Magnetic Resonance Imaging (MRI). Yet, capability of MRI to assess vascular functional reserve and potential of oxygenation-sensitive MRI remain largely unexplored in this model. For this purpose, we compared MC-38 colon and A549 lung adenocarcinoma cell grafts grown on the CAM, using quantitative T1 and T2* MRI readouts as imaging markers. These are associated with vascular functionality and oxygenation status when compared between periods of air and carbogen exposure. Our data show that in A549 lung adenocarcinoma cell grafts T2* values increased significantly upon carbogen exposure (p < 0.004, Wilcoxon test; no change in T1), while MC-38 grafts displayed no changes in T1 and T2*), indicating that the grafts differ in their vascular response. Heterogeneity with regard to T1 and T2* distribution within the grafts was noted. MC-38 grafts displayed larger T1 and T2* in the graft centre, while in A549 they were distributed more towards the graft surface. Finally, qualitative assessment of gadolinium-enhancement suggests that A549 grafts display more prominent enhancement compared to MC-38 grafts. Furthermore, MC-38 grafts had 65% larger volumes than A549 grafts. Histology revealed distinct underlying phenotypes of the two tumor grafts, pertaining to the proliferative status (Ki-67) and cellularity (H&E). In sum, a functional gas challenge with carbogen is feasible through gas exchange on the CAM, and it affects MRI signals associated with vascular reactivity and oxygenation status of the tumor graft planted on the CAM. Different grafts based on A549 lung adenocarcinoma and MC-38 colon carcinoma cell lines, respectively, display distinct phenotypes that can be distinguished and characterized non-invasively in ovo using MRI in the living chicken embryo.

Correction to: protease-activated receptors (PARs): mechanisms of action and potential therapeutic modulators in PAR-driven inflammatory diseases.

[This corrects the article DOI: 10.1186/s12959-019-0194-8.].

High-affinity Cu(I) chelator PSP-2 as potential anti-angiogenic agent.

Copper is an essential trace metal that has been implicated in angiogenesis, the formation of new blood vessels. As tumor growth relies on establishing a functional capillary network for blood supply, copper chelation therapy may hold promise as an anti-cancer strategy by suppressing angiogenesis. To test the anti-angiogenic effect of PSP-2, a recently developed high affinity Cu(I) chelator with low zeptomolar dissociation constant, we utilized the endothelial cancer cell line EAhy926 and assessed changes in cell migration, proliferation, and tube formation in Matrigel. In addition, sprouting was assessed by the chicken and sheep aortic ring assay, and vascular pattern formation was studied in the chorioallantoic membrane of chicken embryos (CAM assay). While incubation with PSP-2 resulted in selective depletion of cellular copper levels, cell migration was not affected and the proliferating activity was even slightly increased. Moreover, the endothelial tube formation assay revealed significant morphological changes in the presence of PSP-2, with thicker tubular walls and an overall decreased meshes area. Similarly, the aortic ring assay and CAM assay showed that PSP-2 evokes significantly longer sprouts with smaller angles at branching points. Altogether, PSP-2 exhibits significant bioactivity at concentrations as low as 5 µM, rendering it a promising anti-angiogenic agent. As EAhy926 cells exhibit both endothelial and tumorigenic properties, the anti-angiogenic effect of PSP-2 might potentially translate also into anti-cancer activity.

Protease-activated receptors (PARs): mechanisms of action and potential therapeutic modulators in PAR-driven inflammatory diseases.

Inflammatory diseases have become increasingly prevalent with industrialization. To address this, numerous anti-inflammatory agents and molecular targets have been considered in clinical trials. Among molecular targets, protease-activated receptors (PARs) are abundantly recognized for their roles in the development of chronic inflammatory diseases. In particular, several inflammatory effects are directly mediated by the sensing of proteolytic activity by PARs. PARs belong to the seven transmembrane domain G protein-coupled receptor family, but are unique in their lack of physiologically soluble ligands. In contrast with classical receptors, PARs are activated by N-terminal proteolytic cleavage. Upon removal of specific N-terminal peptides, the resulting N-termini serve as tethered activation ligands that interact with the extracellular loop 2 domain and initiate receptor signaling. In the classical pathway, activated receptors mediate signaling by recruiting G proteins. However, activation of PARs alternatively lead to the transactivation of and signaling through receptors such as co-localized PARs, ion channels, and toll-like receptors. In this review we consider PARs and their modulators as potential therapeutic agents, and summarize the current understanding of PAR functions from clinical and in vitro studies of PAR-related inflammation.

Thrombin cleaves and activates the protease-activated receptor 2 dependent on thrombomodulin co-receptor availability.

INTRODUCTION: Protease-activated receptors (PARs) evolved to react to extracellular proteolytic activity. In mammals, three of the four PARs (PAR1, PAR3, and PAR4) that are expressed respond to the prototypical procoagulant enzyme thrombin, whereas PAR2 was assumed to resist activation by thrombin. To date, involvement of cell surface thrombin-recruiting co-receptors such as thrombomodulin (TM), which potentially facilitates PAR2 cleavage, has not been addressed. Thus, we examined whether TM-bound thrombin cleaved PAR2 and tested biological responses such as nuclear factor kappa B (NF-κB) DNA binding activity and cytokine release. MATERIALS AND METHODS: We examined 293T cells overexpressing PAR2 and TM for thrombin recruitment by TM promoting PAR2 cleavage. To test for the TM-thrombin interactions required for PAR2 cleavage and to map cleavage sites on PAR2, mutant constructs of TM or PAR2 were engineered. Biological effects because of PAR2 activation were investigated using an NF-κB reporter system and cytokine release. RESULTS AND CONCLUSIONS: We identified that, at low to moderate concentrations, thrombin cleaved PAR2 in a TM co-receptor-dependent manner with cleavage efficiency comparable to that of trypsin. In TM's presence, thrombin efficiently cleaved both, PAR1 and PAR2, albeit kinetics differed. Whereas the majority of surface expressed PAR1 was immediately cleaved off, prolonged exposure to thrombin resulted in few additional cleavage. In contrast, PAR2 cleavage was sustained upon prolonged exposure to thrombin. However, TM EGF-like domain 5 was required and TM chondroitin sulfate (CS) proteoglycan sites serine 490 and serine 492 assisted in PAR2 cleavage, while thrombin preferentially cleaved at arginine 36 on PAR2's N-terminus. Note that thrombin-induced activation of NF-κB via PAR2 resulted in release of interleukin-8. Thus, we provide a novel concept of how thrombin efficiently cleaves PAR2 in a TM-dependent manner, resulting in pro-inflammatory interleukin-8 release. This unexpected pro-inflammatory role of TM, promoting cleavage and activation of PAR2 by thrombin, may lead to novel therapeutic options for treating inflammatory and malignant diseases.