The origin and development of fat droplet and its role in health and illness.

Lipid droplets are found in most cells, where they are needed for lipid metabolism and energy homeostasis. Their biogenesis is still not entirely described process, in which an organelle with monomolecular layer on the surface originates from two-layer membrane of the smooth endoplasmic reticulum. Large lipid droplets can be formed either by growth or by fusion of smaller lipid droplets. Their basic functions are controlled lipid deposition and lipid metabolism and the protection of the intracellular environment from lipotoxicity. Currently, dysfunction of lipid droplet is associated with a number of diseases such as obesity, cardiometabolic syndrome, tumor and neurodegenerative diseases, lipodystrophy and cachexia. Keywords: lipid droplet, adipocyte, endoplasmic reticulum, perilipin, obesity, type 2 diabetes mellitus, lipodystroph.

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.

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.

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.

Bevacizumab Does Not Inhibit the Formation of Liver Vessels and Liver Regeneration Following Major Hepatectomy: A Large Animal Model Study.

BACKGROUND/AIM: Patients with unresectable liver colorectal cancer metastases are treated with neoadjuvant chemotherapy often accompanied by biological therapy aimed at reducing the mass of metastases and thus increasing the chances of resectability. Bevacizumab comprises an anti-VEGF (vascular endothelial growth factor) humanized IgG monoclonal antibody that is used for biological therapy purposes. It acts to inhibit angiogenesis, thereby slowing down the growth of metastases. Due to its being administered systematically, bevacizumab also exerts an effect on the surrounding healthy liver parenchyma and potentially limits the process of neovascularization and thus regeneration of the liver. Since the remnant liver volume forms an important factor in postoperative morbidity and mortality following a major hepatectomy, we decided to study the effect of bevacizumab on vascular and biliary microarchitecture in healthy liver parenchyma and its ability to regenerate following major hepatectomy. MATERIALS AND METHODS: We performed an experiment employing a large animal model where a total of 16 piglets were divided into two groups (8 piglets in the control group and 8 piglets in the experimental group with bevacizumab). All the animals were subjected to major hepatectomy and the experimental group was given bevacizumab prior to hepatectomy. All the animals were sacrificed after 4 weeks. We performed biochemical analyses at regular time intervals during the follow-up period. Histological examination of the liver tissue was performed following sacrifice of the animals. RESULTS: No statistical difference was shown between groups in terms of the biochemical and immunohistochemical parameters. The histological examination of the regenerating liver tissue revealed the higher length density of sinusoids in the experimental group. CONCLUSION: Bevacizumab does not act to impair liver regeneration following hepatectomy.

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.

Blunt injury of liver: mechanical response of porcine liver in experimental impact test.

OBJECTIVE: The liver is frequently injured in blunt abdominal trauma caused by road traffic accidents. The testing of safety performance of vehicles, e.g. belt usage, head support, seat shape, or air bag shape, material, pressure and reaction, could lead to reduction of the injury seriousness. Current trends in safety testing include development of accurate computational human body models (HBMs) based on the anatomical, morphological, and mechanical behavior of tissues under high strain. APPROACH: The aim of this study was to describe the internal pressure changes within porcine liver, the severity of liver injury and the relation between the porcine liver microstructure and rupture propagation in an experimental impact test. Porcine liver specimens (n = 24) were uniformly compressed using a drop tower technique and four impact heights (200, 300, 400 and 500 mm; corresponding velocities: 1.72, 2.17, 2.54 and 2.88 m s-1). The changes in intravascular pressure were measured via catheters placed in portal vein and caudate vena cava. The induced injuries were analyzed on the macroscopic level according to AAST grade and AIS severity. Rupture propagation with respect to liver microstructure was analyzed using stereological methods. MAIN RESULTS: Macroscopic ruptures affected mostly the interface between connective tissue surrounding big vessels and liver parenchyma. Histological analysis revealed that the ruptures avoided reticular fibers and interlobular septa made of connective tissue on the microscopic level. SIGNIFICANCE: The present findings can be used for evaluation of HBMs of liver behavior in impact situations.

Hydrogel Containing Anti-CD44-Labeled Microparticles, Guide Bone Tissue Formation in Osteochondral Defects in Rabbits.

Hydrogels are suitable for osteochondral defect regeneration as they mimic the viscoelastic environment of cartilage. However, their biomechanical properties are not sufficient to withstand high mechanical forces. Therefore, we have prepared electrospun poly-ε-caprolactone-chitosan (PCL-chit) and poly(ethylene oxide)-chitosan (PEO-chit) nanofibers, and FTIR analysis confirmed successful blending of chitosan with other polymers. The biocompatibility of PCL-chit and PEO-chit scaffolds was tested; fibrochondrocytes and chondrocytes seeded on PCL-chit showed superior metabolic activity. The PCL-chit nanofibers were cryogenically grinded into microparticles (mean size of about 500 µm) and further modified by polyethylene glycol-biotin in order to bind the anti-CD44 antibody, a glycoprotein interacting with hyaluronic acid (PCL-chit-PEGb-antiCD44). The PCL-chit or PCL-chit-PEGb-antiCD44 microparticles were mixed with a composite gel (collagen/fibrin/platelet rich plasma) to improve its biomechanical properties. The storage modulus was higher in the composite gel with microparticles compared to fibrin. The Eloss of the composite gel and fibrin was higher than that of the composite gel with microparticles. The composite gel either with or without microparticles was further tested in vivo in a model of osteochondral defects in rabbits. PCL-chit-PEGb-antiCD44 significantly enhanced osteogenic regeneration, mainly by desmogenous ossification, but decreased chondrogenic differentiation in the defects. PCL-chit-PEGb showed a more homogeneous distribution of hyaline cartilage and enhanced hyaline cartilage differentiation.

Influence of Mesenchymal Stem Cell Administration on The Outcome of Partial Liver Resection in a Porcine Model of Sinusoidal Obstruction Syndrome.

BACKGROUND: In patients with colorectal liver metastases, the possibility for radical liver resection can be limited by oxaliplatin-induced sinusoidal obstruction syndrome (SOS). This study investigates the potential of mesenchymal stem cells (MSC) to improve the outcome of liver resections in pigs with SOS. MATERIALS AND METHODS: SOS was induced in all animals (n=20) on day 0. Animals in the experimental group (n=8) received allogeneic MSC on day 7. Liver resection was performed in all animals on day 14 and the animals were observed until day 28. Ultrasound volumetry, biochemical analysis and histological examination of liver parenchyma was performed during the follow-up period. RESULTS: Six animals from the control group died prematurely, while all animals survived in the experimental group. According to histology, biochemical analysis and ultrasound volumetry, there were no significant differences between the groups documenting the effect of MSC. CONCLUSION: Single dose allogeneic MSC administration improved survival of animals with SOS undergoing partial liver resection. Further experiments with different timing of liver resection and MSC administration should be performed to investigate the effect of MSC in more detail.

Alopecia and Hair Damage Induced by Oncological Therapy.

Damage and loss of hair (alopecia) is a predictable adverse event of oncological therapy. It can be caused by chemotherapy, radiotherapy, or targeted and hormonal therapy. From the point of view of patients with malignant disease, hair loss is one of the most feared side effects and adversely affects their mental health. Hair loss can be diffuse, complete, partial, or regional. Worsening of hair quality, cutaneous inflammation, and scarring can also occur. Eyelashes, eyebrows, and body hair can also be lost. Alopecia is mostly reversible, but permanent damage can occur depending on the type, overall length, and dose of oncological treatment and other factors. The risk of alopecia is high with high-dose docetaxel, doxorubicin, and cyclophosphamide, but low with platinum chemotherapy, melphalan, and capecitabin. Targeted therapy and immunotherapy can cause immune-mediated alopecia such as alopecia areata and scarring alopecia as well as paradoxically hypertrichosis and trichomegaly. Physical and pharmacological approaches can be used to prevent and treat alopecia; however, their effectiveness and availability are limited. Modern radiotherapy scalp-sparing methods minimize hair loss. Good results have been obtained with scalp cooling, which reduces the toxic effects of cytostatic agents on hair follicles during short infusion regimens. Several systems cool the scalp to less than 22°C. Minoxidil accelerates hair regrowth and is used as a topical therapy. Psychological support and provision of cosmetically acceptable head coverings are also very important.

Generating standardized image data for testing and calibrating quantification of volumes, surfaces, lengths, and object counts in fibrous and porous materials using X-ray microtomography.

Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans.