Periphery of porcine hepatic lobes has the smallest length density of hepatic sinusoids and bile canaliculi: A stereological histological study with implications for liver biopsies.

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.

Histological mapping of healing of the small and large intestine - A quantitative study in a porcine model.

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.

Nanofiber Fractionalization Stimulates Healing of Large Intestine Anastomoses in Rabbits.

Background: A current topic of ma jor interest in regenerative medicine is the development of novel materials for accelerated healing of sutures, and nanofibers seem to be suitable materials for this purpose. As various studies have shown, nanofibers are able to partially substitute missing extracellular matrix and to stimulate cell proliferation and differentiation in sutures. Therefore, we tested nanofibrous membranes and cryogenically fractionalized nanofibers as potential materials for support of the healing of intestinal anastomoses in a rabbit model. Materials and Methods: We compared cryogenically fractionalized chitosan and PVA nanofibers with chitosan and PVA nanofiber membranes designed for intestine anastomosis healing in a rabbit animal model. The anastomoses were biomechanically and histologically tested. Results: In strong contrast to nanofibrous membranes, the fractionalized nanofibers did show positive effects on the healing of intestinal anastomoses in rabbits. The fractionalized nanofibers were able to reach deep layers that are key to increased mechanical strength of the intestine. Moreover, fractionalized nanofibers led to the formation of collagen-rich 3D tissue significantly exceeding the healing effects of the 2D flat nanofiber membranes. In addition, the fractionalized chitosan nanofibers eliminated peritonitis, significantly stimulated anastomosis healing and led to a higher density of microvessels, in addition to a larger fraction of myofibroblasts and collagen type I and III. Biomechanical tests supported these histological findings. Conclusion: We concluded that the fractionalized chitosan nanofibers led to accelerated healing for rabbit colorectal anastomoses by the targeted stimulation of collagen-producing cells in the intestine, the smooth muscle cells and the fibroblasts. We believe that the collagen-producing cells were stimulated both directly due to the presence of a biocompatible scaffold providing cell adhesion, and indirectly, by a proper stimulation of immunocytes in the suture.

Porcine spleen as a model organ for blunt injury impact tests: An experimental and histological study.

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.

Complex genetic and histopathological study of 15 patient-derived xenografts of aggressive lymphomas.

Non-Hodgkin lymphomas (NHL) represent the most common hematologic malignancies. Patient-derived xenografts (PDXs) are used for various aspects of translational research including preclinical in vivo validation of experimental treatment approaches. While it was repeatedly demonstrated that PDXs keep majority of somatic mutations with the primary lymphoma samples, from which they were derived, the composition of PDX tumor microenvironment (TME) has not been extensively studied. We carried out a comparative genetic and histopathological study of 15 PDX models derived from patients with various types of NHL including diffuse large B-cell lymphoma (DLBCL; n = 7), Burkitt lymphoma (BL; n = 1), mantle cell lymphoma (MCL; n = 2), and peripheral T-cell lymphomas (PTCL; n = 5). Whole exome sequencing (WES) of the PDXs and primary lymphoma cells was implemented in 13 out of 15 cases with available DNA samples. Standard immunohistochemistry (IHC) was used to analyze the composition of PDX TME. WES data confirmed that PDXs maintained the genetic heterogeneity with the original primary lymphoma cells. In contrast, IHC analysis revealed the following recurrently observed alterations in the composition of PDX tumors: more blastoid lymphoma cell morphology, increased proliferation rate, lack of non-malignant cellular components including T cells and (human or murine) macrophages, and significantly lower intratumoral microvessel density and microvessel area composed of murine vessels. In addition, PDX tumors derived from T-NHL displayed additional differences compared to the primary lymphoma samples including markedly lower desmoplasia (i.e., the extent of both reticular and collagen fibrosis), loss of expression of cytotoxic granules (i.e., perforin, TIA, granzyme B), or loss of expression of T-cell specific antigens (i.e., CD3, CD4, CD8). Our data suggest that despite keeping the same genetic profiles, PDX models of aggressive NHL do not recapitulate the microenvironmental heterogeneity of the original lymphomas. These findings have implications on the relevance of PDX models in the context of preclinical research.

Are ovine and porcine carotid arteries equivalent animal models for experimental cardiac surgery: A quantitative histological comparison.

BACKGROUND: Coronary artery bypass grafting (CABG) is a common cardiac surgery. Manufacturing small-diameter (2-5 mm) vascular grafts for CABG is important for patients who lack first-choice autologous arterial, or venous conduits. Ovine and porcine common carotid arteries (CCAs) are used as large animal models for in vivo testing of newly developed tissue-engineered arterial grafts. It is unknown to what extent these models are interchangeable and whether the left and right arteries of the same subjects can be used as experimental controls. Therefore, we compared the microscopic structure of paired left and right ovine and porcine CCAs in the proximodistal direction and compared these animal model samples to samples of human coronary arteries (CAs) and human internal thoracic arteries (ITAs). METHODS: We compared the histological composition of whole CCAs of sheep (n = 22 animals) with whole porcine CCAs (n = 21), segments of human CAs (n = 21), and human ITAs (n = 21). Using unbiased sampling and stereological methods, we quantified the fractions of elastin, total collagen, type I collagen, type III collagen, smooth muscle actin (SMA) and chondroitin sulfate (CS) A, B, and C. We also quantified the densities and distributions of nuclear profiles, nervi vasorum and vasa vasorum as well as the thickness of the intima-media and total wall thickness. RESULTS: The differences between the paired samples of left and right CCAs in sheep were substantially greater than the differences in laterality in porcine CCAs. The right ovine CCAs had a smaller fraction of elastin (p < 0.001), greater fraction of SMA (p < 0.01), and greater intima-media thickness (p < 0.001) than the paired left side CCAs. In pigs, the right CCAs had a greater fraction of elastin (p < 0.05) and a greater density of vasa vasorum in the media (p < 0.001) than the left-side CCAs. The fractions of elastin and CS decreased and the fraction of SMA increased in the proximodistal direction in both the ovine (p < 0.001) and porcine (p < 0.001) CCAs. Ovine CCAs had a muscular phenotype along their entire length, but porcine CCAs were elastic-type arteries in the proximal segments but muscular type arteries in middle and distal segments. The CCAs of both animals differed from the human CAs and ITAs in most parameters, but the ovine CCAs had a comparable fraction of elastin and CS to human ITAs. CONCLUSIONS: From a histological point of view, ovine and porcine CCAs were not equivalent in most quantitative parameters to human CAs and ITAs. Left and right ovine CCAs did not have the same histological composition, which is limiting for their mutual equivalence as sham-operated controls in experiments. These differences should be taken into account when designing and interpreting experiments using these models in cardiac surgery. The complete morphometric data obtained by quantitative evaluation of arterial segments were provided to facilitate the power analysis necessary for justification of the minimum number of samples when planning further experiments. The middle or distal segments of ovine and porcine CCAs remain the most realistic and the best characterized large animal models for testing artificial arterial CABG conduits.

Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Quantitative In Vivo Monitoring of Hypoxia and Vascularization of Patient-Derived Murine Xenografts of Mantle Cell Lymphoma Using Photoacoustic and Ultrasound Imaging.

Tumor oxygenation and vascularization are important parameters that determine the aggressiveness of the tumor and its resistance to cancer therapies. We introduce dual-modality ultrasound and photoacoustic imaging (US-PAI) for the direct, non-invasive real-time in vivo evaluation of oxygenation and vascularization of patient-derived xenografts (PDXs) of B-cell mantle cell lymphomas. The different optical properties of oxyhemoglobin and deoxyhemoglobin make it possible to determine oxygen saturation (sO2) in tissues using PAI. High-frequency color Doppler imaging enables the visualization of blood flow with high resolution. Tumor oxygenation and vascularization were studied in vivo during the growth of three different subcutaneously implanted patient-derived xenograft (PDX) lymphomas (VFN-M1, VFN-M2 and VFN-M5 R1). Similar values of sO2 (sO2 Vital), determined from US-PAI volumetric analysis, were obtained in small and large VFN-M1 tumors ranging from 37.9 ± 2.2 to 40.5 ± 6.0 sO2 Vital (%) and 37.5 ± 4.0 to 35.7 ± 4.6 sO2 Vital (%) for small and large VFN-M2 PDXs. In contrast, the higher sO2 Vital values ranging from 57.1 ± 4.8 to 40.8 ± 5.7 sO2 Vital (%) (small to large) of VFN-M5 R1 tumors corresponds with the higher aggressiveness of that PDX model. The different tumor percentage vascularization (assessed as micro-vessel areas) of VFN-M1, VFN-M2 and VFN-M5 R1 obtained by color Doppler (2.8 ± 0.1%, 3.8 ± 0.8% and 10.3 ± 2.7%) in large-stage tumors clearly corresponds with their diverse growth and aggressiveness. The data obtained by color Doppler were validated by histology. In conclusion, US-PAI rapidly and accurately provided relevant and reproducible information on tissue oxygenation in PDX tumors in real time without the need for a contrast agent.

Histological mapping of porcine carotid arteries - An animal model for the assessment of artificial conduits suitable for coronary bypass grafting in humans.

BACKGROUND: Using animal models in experimental medicine requires mapping of their anatomical variability. Porcine common carotid arteries (CCA) are often preferred for the preclinical testing of vascular grafts due to their anatomical and physiological similarity to human small-diameter arteries. Comparing the microscopic structure of animal model organs to their human counterparts reveals the benefits and limitations of translational medicine. METHODS: Using quantitative histology and stereology, we performed an extensive mapping of the regional proximodistal differences in the fractions of elastin, collagen, and smooth muscle actin as well as the intima-media and wall thicknesses among 404 segments (every 1 cm) of porcine CCAs collected from male and female pigs (n = 21). We also compared the microscopic structure of porcine CCAs with segments of human coronary arteries and one of the preferred arterial conduits used for the coronary artery bypass grafting (CABG), namely, the internal thoracic artery (ITA) (n = 21 human cadavers). RESULTS: The results showed that the histological structure of left and right porcine CCA can be considered equivalent, provided that gross anatomical variations of the regular branching patterns are excluded. The proximal elastic carotid (51.2% elastin, 4.2% collagen, and 37.2% actin) transitioned to more muscular middle segments (23.5% elastin, 4.9% collagen, 54.3% actin) at the range of 2-3 centimeters and then to even more muscular distal segments (17.2% elastin, 4.9% collagen, 64.0% actin). The resulting morphometric data set shows the biological variability of the artery and is made available for biomechanical modeling and for performing a power analysis and calculating the minimum number of samples per group when planning further experiments with this widely used large animal model. CONCLUSIONS: Comparison of porcine carotids with human coronary arteries and ITA revealed the benefits and the limitations of using porcine CCAs as a valid model for testing bioengineered small-diameter CABG vascular conduits. Morphometry of human coronary arteries and ITA provided more realistic data for tailoring multilayered artificial vascular prostheses and the ranges of values within which the conduits should be tested in the future. Despite their limitations, porcine CCAs remain a widely used and well-characterized large animal model that is available for a variety of experiments in vascular surgery.