There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The prognostic significance of mast cells and different phenotypes of macrophages in the microenvironment of hepatocellular carcinoma (HCC) following resection is unclear. We aimed in this study to assess the local distribution of infiltrating macrophages and mast cells of specific phenotypes in tissues of HCC and to evaluate their prognostic values for survival of post-surgical patients. METHODS: The clinicopathological and follow-up data of 70 patients with HCC, who underwent curative resection of tumor from 1997 to 2019, were collected. The infiltration of CD68+ and CD163+ macrophages and CD117+ mast cells was assessed immunohistochemically in representative resected specimens of HCC and adjacent tissues. The area fraction (AF) of positively stained cells was estimated automatically using QuPath image analysis software in several regions, such as tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells, individually and in associations, for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: High AF of CD68+ macrophages in TC and IM and high AF of mast cells in IM and PT area were associated with a longer DFS. High AF of CD163+ macrophages in PT area correlated with a shorter DFS. Patients from CD163TChigh & CD68TClow group had a shorter DFS compared to all the rest of the groups, and cases with CD163IMlow & CD68IMhigh demonstrated significantly longer DFS compared to low AF of both markers. Patients from CD68IMhigh & CD163PTlow group, CD117IMhigh & CD163PTlow group, and CD117PThigh & CD163PTlow group had a significantly longer DFS compared to all other combinations of respective cells. CONCLUSIONS: The individual prognostic impact of CD68+ and CD163+ macrophages and mast cells in the microenvironment of HCC after resection depends on their abundance and location, whereas the cumulative impact is built upon combination of different cell phenotypes within and between regions.
BACKGROUND: ATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients' prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers - ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Protein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. RESULTS: No CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). CONCLUSIONS: The study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.
- MeSH
- ABC transportéry genetika MeSH
- adenokarcinom * patologie MeSH
- duktální karcinom pankreatu * patologie MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- nádory slinivky břišní * diagnóza patologie MeSH
- prognóza MeSH
- progrese nemoci MeSH
- receptory sulfonylurey * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Porcine liver is widely used in hepatologic research as a large animal model with many anatomical and physiological similarities with humans. However, only limited information on porcine liver spatial microstructure has been published, especially regarding the hepatic sinusoids and bile canaliculi. The aim of our study was to quantify the sinusoidal and bile canalicular network in healthy male and female porcine livers and to map the variability of these structures with heterogenous distribution to improve the evaluability of liver biopsy samples. METHODS: Livers from 12 healthy piglets (6 females and 6 neutered males) were sampled into 36 tissue samples per organ, representing six hepatic lobes and three different regions related to the hepatic vasculature (peripheral, paracaval and paraportal region). Histological sections were processed with a random orientation of the cutting plane. The endothelium and the bile canaliculi were stained using Ricinus communis agglutinin I lectin histochemistry. The length densities of hepatic sinusoids LV(sinusoids,liver), of bile canaliculi LV(bile canaliculi,liver) and volume fraction VV(sinusoids,liver) and surface density SV(sinusoids,liver) of sinusoids were estimated using stereological methods. The newly acquired morphometric data were compared with previously published data on density of porcine hepatocytes and fractions of connective tissue. RESULTS: The peripheral region had smallest LV(sinusoids,liver), smallest LV(bile canaliculi,liver) and greatest VV(sinusoids,liver). The six hepatic lobes had statistically comparable length densities of both sinusoids and bile canaliculi, but the left lateral lobe had smallest VV(sinusoids,liver). Regions with greater LV(sinusoids,liver) had also greater LV(bile canaliculi,liver) and SV(sinusoids,liver) and were accompanied by greater density of smaller hepatocytes. Regions with smaller LV(sinusoids,liver) and LV(bile canaliculi,liver) contained a greater fraction of interlobular connective tissue. CONCLUSIONS: The length density of hepatic sinusoids is smaller in the peripheral regions of the porcine liver than in other regions related to the hepatic vasculature - paracaval and paraportal regions, and smaller in castrated males than in females. Greater length density of liver sinusoids was linked with greater local density of bile canaliculi, with local increase in the density of smaller hepatocytes and, simultaneously, with smaller fractions of hepatic connective tissue. The intrahepatic and inter-sexual variability of the porcine liver morphology needs to be taken into account when designing and interpreting experiments involving the histological quantification of the microvascular network. The complete primary morphometric data describing the distribution of morphometric parameters within porcine liver were made available in a form facilitating the power analysis to justify the minimal number of tissue samples or animals required when designing further histological evaluation studies. The macroscopic map of microvessels and bile canaliculi variability facilitates their assessment in liver biopsies in the pig.
- MeSH
- biopsie MeSH
- hepatocyty MeSH
- játra anatomie a histologie MeSH
- kapiláry * MeSH
- lidé MeSH
- prasata MeSH
- žlučové kanálky * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Anastomotický leak je v kolorektální chirurgii závažnou komplikací. Problematikou poruch hojení střevní anastomózy se zabývá řada experimentálních prací zejména ve smyslu aplikovaného výzkumu. Design zvířecích modelů je přitom různorodý a výsledky jednotlivých prací jsou těžko porovnatelné. Tato práce pojednává souhrnně o hlavních bodech problematiky plánování zvířecích modelů střevních anastomóz, které jsou jednotlivě rozebrány. Hlavní částí textu je popis modelu defektní střevní anastomózy prasete. Anastomóza je v modelu konstruována s lokalizovanou protruzí sliznice. Zvířata jsou pooperačně sledována 3 týdny, monitorovány jsou jak klinický stav, tak změny vitálních hodnot, laboratorních parametrů, je prováděno v definovaných bodech CT vyšetření. Na konci observačního období jsou odebrány vzorky, hodnocen makroskopický nález v dutině břišní, skórování adhezí, známky leaku či poruch pasáže. Histologicky jsou preparáty hodnoceny jak standardními metodami analyzujícími vaskularitu, zánětlivou infiltraci a podíl kolagenu, tak metodami vyvinutými de novo pro potřeby experimentu, jako je analýza integrity střevní stěny v místě slizniční protruze. Experimentálního modelu si ceníme pro možnost systematické a podrobné analýzy stavu zhojení anastomózy v kombinaci s podrobným observačním protokolem, jež vytvářejí klinicky relevantní výsledky.
Anastomotic leak is a serious complication in colorectal surgery. The problem of intestinal anastomosis healing disorders is dealt with by a number of experimental studies, especially in applied research. The design of animal models is diverse and the results of individual studies are difficult to compare. This paper summarizes the main issues of planning animal models of intestinal anastomoses, which are discussed individually. The main part of the text is a description of the defective intestinal anastomosis model of the pig. The anastomosis is constructed in the model with a localized mucosal protrusion. The animals are monitored for 3 weeks postoperatively, the clinical condition and changes in vital values and laboratory parameters are monitored, and CT examinations are performed at defined points. At the end of the observation period, samples are taken, macroscopic findings in the abdominal cavity are evaluated, adhesions are scored, and signs of leakage or passage disorders are assessed. The preparations are evaluated histologically both by standard methods analyzing vascularity, inflammatory infiltration and the proportion of collagen, and by methods developed de novo for the needs of the experiment, such as the analysis of the integrity of the intestinal wall at the site of the mucosal protrusion. We value the experimental model for the possibility of a systematic and detailed analysis of the healing state of the anastomosis in combination with a detailed observation protocol, which produces clinically relevant results.
BACKGROUND: Gastrointestinal anastomoses are performed in many patients every year. The pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage are not fully understood. The present study gathered and critically evaluated histological quantitative data to deepen current knowledge of anastomotic healing in the small and large intestine and its complications and outline the options for further experimental in vivo research in large porcine animal models. METHODS: Three groups of porcine intestinal anastomoses were compared: small intestine without defect (SI; n = 7), small intestine with an additional defect (SID; n = 8), and large intestine (LI; n = 7). Multilevel sampling (2112 micrographs) and stereological methods were used for histological quantification of proliferation (Ki-67 immunohistochemistry), neutrophil infiltration (myeloperoxidase staining), vascularity (von Willebrand factor) and type I and type III collagen formation (picrosirius red in polarized light) within the region of anastomosis compared to the region outside of anastomosis. RESULTS: Quantitative histological evaluation revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluation of surgical experiments. The presence or absence of an additional experimental defect strongly affected healing, but the healing seemed complete after 21 days. iii) The microscopic structure of small intestine segments was more affected by their proximity to the anastomosis than the structure of large intestine segments. CONCLUSIONS: Histological quantification was more laborious than the previously used semiquantitative scoring system evaluating the healing rate of intestinal anastomoses, but it provided detailed maps of biological processes within individual intestine layers. The primary data collected in the study are open and available for power sample analyses to calculate the minimum numbers of samples justified in future experiments on porcine intestines. The porcine intestine is a promising animal model with translational potential for human surgery.
- MeSH
- anastomóza chirurgická metody MeSH
- hojení ran * MeSH
- lidé MeSH
- prasata MeSH
- střeva MeSH
- tenké střevo * MeSH
- tlusté střevo MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Hepatocellular carcinoma (HCC) is a fatal disease characterized by early genetic alterations in telomerase reverse transcriptase promoter (TERTp) and β-catenin (CTNNB1) genes and immune cell activation in the tumor microenvironment. As a novel approach, we wanted to assess patient survival influenced by combined presence of mutations and densities of CD8+ cytotoxic T cells. METHODS: Tissue samples were obtained from 67 HCC patients who had undergone resection. We analysed CD8+ T cells density, TERTp mutations, rs2853669 polymorphism, and CTNNB1 mutations. These variables were evaluated for time to recurrence (TTR) and disease free survival (DFS). RESULTS: TERTp mutations were found in 75.8% and CTNNB1 mutations in 35.6% of the patients. TERTp mutations were not associated with survival but polymorphism rs2853669 in TERTp was associated with improved TTR and DFS. CTNNB1 mutations were associated with improving TTR. High density of CD8+ T-lymphocytes in tumor center and invasive margin correlated with longer TTR and DFS. Combined genetic and immune factors further improved survival showing higher predictive values. E.g., combining CTNNB1 mutations and high density of CD8+ T-lymphocytes in tumor center yielded HRs of 0.12 (0.03-0.52), p = 0.005 for TTR and 0.25 (0.09-0.74), p = 0.01 for DFS. CONCLUSION: The results outline a novel integrative approach for prognostication through combining independent predictive factors from genetic and immune cell profiles. However, larger studies are needed to explore multiple cell types in the tumor microenvironment.
- MeSH
- beta-katenin genetika MeSH
- CD8-pozitivní T-lymfocyty patologie MeSH
- hepatocelulární karcinom * genetika patologie chirurgie MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- mutace MeSH
- nádorové mikroprostředí genetika MeSH
- nádory jater * genetika patologie chirurgie MeSH
- počet buněk MeSH
- telomerasa * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The spleen is a large and highly vascularized secondary lymphatic organ. Spleen injuries are among the most frequent trauma-related injuries in the abdominal region. The aims of the study were to assess the volume fractions of the main splenic tissue components (red pulp, white pulp, trabeculae and reticular fibres) and to determine the severity of splenic injury due to the experimental impact test. Porcine spleens (n = 17) were compressed by 6.22 kg wooden plate using a drop tower technique from three impact heights (50, 100 and 150 mm corresponding to velocities 0.79, 1.24 and 1.58 m/s). The pressure was measured via catheters placed in the splenic vein. The impact velocity was measured using lasers. The severity of induced injuries was analysed on the macroscopic level. The volume fractions of splenic components were assessed microscopically using stereology. The volume fraction of the red pulp was 76.4%, white pulp 21.3% and trabeculae 2.7% respectively. All impact tests, even with the low impact velocities, led to injuries that occurred mostly in the dorsal extremity of the spleen, and were accompanied by bleeding, capsule rupture and parenchyma crushing. Higher impact height (impact velocity and impact energy) caused more severe injury. Porcine spleen had the same volume fraction of tissue components as human spleen, therefore we concluded that the porcine spleen was a suitable organ model for mechanical experiments. Based on our observations, regions around hilum and the diaphragmatic surface of the dorsal extremity, that contained fissures and notches, were the most prone to injury and required considerable attention during splenic examination after injury. The primary mechanical data are now available for the researchers focused on the splenic trauma modelling.
- MeSH
- lidé MeSH
- nemoci prasat * MeSH
- prasata MeSH
- slezina MeSH
- tupá poranění * veterinární MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.
- Publikační typ
- časopisecké články MeSH
Úvod: Při využití prasete jako experimentálního zvířete je často nezbytné zajištění permanentního žilního vstupu, který umožní nejen opakované krevní odběry. Cílem práce bylo vyhodnotit průchodnost a komplikace implantabilních intravenózních port katétrů u prasat zařazených do různých chirurgických experimentů. Metody: Port katétry byly implantovány cestou zevní jugulární žíly celkem 211 prasatům zařazeným do 7 různých experimentů. Retrospektivně byly hodnoceny všechny zaznamenané komplikace. Výsledky: U 157 zvířat (74,4 %) nebyly zaznamenány žádné komplikace. Nejméně závažnou komplikací byl otok (12 zvířat, 5,7 %) a serom v okolí portu (3 zvířata, 1,4 %). Přechodné potíže s aspirací krve byly zaznamenány u 13 zvířat (6,2 %). Nejzávažnější komplikace, kvůli kterým nemohl být port katétr využíván k aspiraci ani aplikaci, nastaly u 26 zvířat (12,3 %). Jednalo se o absces v okolí portu (12 zvířat), nekrózu kožního krytu (2 zvířata), částečnou dehiscenci rány (2 zvířata) a ztrátu funkce port katétru z nespecifikovaných důvodů (10 zvířat). U žádného ze zvířat nemusel být port katétr explantován a žádné zvíře nemuselo být z důvodů komplikací port katétru vyřazeno z experimentu. Cestou port katétru lze bezpečně aplikovat i jodovou kontrastní látku při CT vyšetření. Závěr: I přes zaznamenané komplikace je implantabilní port katétr vhodný k zajištění permanentního intravenózního vstupu u prasat v rámci chirurgických experimentů. Tato metodika umožňuje minimalizovat stres zvířat v pooperačním období a redukovat jejich počet.
Introduction: Permanent intravenous access is usually required in pigs used for surgical experiments, not only to enable repeated blood sample collections. The aim of this study was to evaluate the patency and complications of vascular access ports (VAP) implanted in pigs included in different surgical experiments. Methods: VAPs were implanted via the external jugular vein in a total of 211 pigs from 7 different experiments. All observed complications were retrospectively evaluated. Results: No complications were observed in 157 animals (74.4%). Complications of the least severity were edema or seroma around the port which were observed in 12 (5.7%) and 3 (1.4%) animals, respectively. Temporary problems with aspiration of blood via the port occurred in 13 animals (6.2%). The most severe complications which prevented the use of the VAP for aspiration and application were recorded in 26 animals (12.3%). These complications included: abscess formation around the port (12 animals), skin necrosis over the port (2 animals), partial wound dehiscence (2 animals) and loss of the VAP function due to an unspecified cause (10 animals). Removal of the VAP was not needed in any of the animals and none of the animals had to be excluded from the experiment due to the complications. The VAP can also be used for safe administration of iodine contrast agent during CT examination. Conclusion: Despite the observed complications the VAP is suitable as permanent intravenous access in pigs used for surgical experiments. This method helps to minimize the stress of the animals in the postoperative period and to reduce the number of experimental animals.
- MeSH
- modely u zvířat MeSH
- prasata MeSH
- zaváděcí katétry * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
