Quantitative Histological Analysis Research Articles

Viscoelastic properties of colorectal liver metastases reflect tumour cell viability

BackgroundColorectal cancer is the third most common tumour entity in the world and up to 50% of the patients develop liver metastases (CRLM) within five years. To improve and personalize therapeutic strategies, new diagnostic tools are urgently needed. For instance, biomechanical tumour properties measured by magnetic resonance elastography (MRE) could be implemented as such a diagnostic tool. We postulate that ex vivo MRE combined with histological and radiological evaluation of CRLM could provide biomechanics-based diagnostic markers for cell viability in tumours.Methods34 CRLM specimens from patients who had undergone hepatic resection were studied using ex vivo MRE in a frequency range from 500 Hz to 5300 Hz with increments of 400 Hz. Single frequency evaluation of shear wave speed and wave penetration rate as proxies for stiffness and viscosity was performed, along with rheological model fitting based on the spring-pot model and powerlaw exponent α, ranging between 0 (complete solid behaviour) and 1 (complete fluid behaviour). For histological analysis, samples were stained with H&E and categorized according to the degree of regression. Quantitative histologic analysis was performed to analyse nucleus size, aspect ratio, and density. Radiological response was assessed according to RECIST-criteria.ResultsFive samples showed major response to chemotherapy, six samples partial response and 23 samples no response. For higher frequencies (> 2100 Hz), shear wave speed correlated significantly with the degree of regression (p ≤ 0.05) indicating stiffer properties with less viable tumour cells. Correspondingly, rheological analysis of α revealed more elastic-solid tissue properties at low cell viability and major response (α = 0.43 IQR 0.36, 0.47) than at higher cell viability and no response (α = 0.51 IQR 0.48, 0.55; p = 0.03). Quantitative histological analysis showed a decreased nuclear area and density as well as a higher nuclear aspect ratio in patients with major response to treatment compared to patients with no response (all p < 0.05).DiscussionOur results suggest that MRE could be useful in the characterization of biomechanical property changes associated with cell viability in CRLM. In the future, MRE could be applied in clinical diagnosis to support individually tailored therapy plans for patients with CRLM.

Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2

Coronavirus disease 2019 (COVID-19) is an immune-related disorder caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The complete pathogenesis of the virus remains to be determined. Unraveling the molecular mechanisms governing SARS-CoV-2 interactions with host cells is crucial for the formulation of effective prophylactic measures and the advancement of COVID-19 therapeutics. We analyzed human lung single-cell RNA sequencing dataset to discern the association of butyrophilin subfamily 3 member A2 (BTN3A2) expression with COVID-19. The BTN3A2 gene edited cell lines and transgenic mice were infected by live SARS-CoV-2 in a biosafety level 3 (BSL-3) laboratory. Immunoprecipitation, flow cytometry, biolayer interferometry and competition ELISA assays were performed in BTN3A2 gene edited cells. We performed quantitative real-time PCR, histological and/or immunohistochemical analyses for tissue samples from mice with or without SARS-CoV-2 infection. The BTN3A2 mRNA level was correlated with COVID-19 severity. BTN3A2 expression was predominantly identified in epithelial cells, elevated in pathological epithelial cells from COVID-19 patients and co-occurred with ACE2 expression in the same lung cell subtypes. BTN3A2 targeted the early stage of the viral life cycle by inhibiting SARS-CoV-2 attachment through interactions with the receptor-binding domain (RBD) of the Spike protein and ACE2. BTN3A2 inhibited ACE2-mediated SARS-CoV-2 infection by reducing ACE2 invitro and invivo. These results reveal a key role of BTN3A2 in the fight against COVID-19. Identifying potential monoclonal antibodies which mimic BTN3A2 may facilitate disruption of SARS-CoV-2 infection, providing a therapeutic avenue for COVID-19. This study was supported by the National Natural Science Foundation of China (32070569, U1902215, and 32371017), the CAS "Light of West China" Program, and Yunnan Province (202305AH340006).

Reproductive cycle, fecundity and growth of the freshwater mussel Unio tumidus (Bivalvia: Unionidae) from Lake Viinijärvi, Finland

ABSTRACT Freshwater mussels have a substantial role in aquatic ecosystem function and provide valuable ecosystem services, including water filtration, nutrient cycling, habitat creation and sediment stabilization. While they face population declines globally that can negatively affect an ecosystem's health, such as reducing the water quality and increasing the turbidity, lack of information about the basic reproductive biology for most species makes their conservation much more difficult. Here, we monitored a population of Unio tumidus for a year to study its reproductive biology, gonad-dwelling parasites and growth rate in Lake Viinijärvi, Finland. Our data revealed that this species was dioecious with no evidence of hermaphroditism and was non-significantly female-biased (a male to female ratio of 1:1.3). Both sexes of U. tumidus had continuous gametogenesis during the study period with two peaks in the gonadosomatic index (GSI%) during November and May, followed by two clear declines in December and June. Gravidity period (brooding) of embryos or glochidia in the female's outer gills lasted from June to July. Therefore, this population of U. tumidus can be classified as a short-term breeder (tachytictic). The clear decline of GSI% in December might be a strategy that individuals of U. tumidus use to resorb resources back from the gonad for somatic maintenance during winter. Mean length of glochidia was 391 ± 0.049 µm, being among the largest known glochidia for Unionidae. No gonad-dwelling trematode parasites were detected and the population exhibited high fecundity (numbers of oocytes per female), with an average oocyte production of 242,000 ± 18,000. The studied population had a moderate growth rate with a maximum predicted age of 13 years and asymptotic length (the length at which growth slows down while the mussel continues to age, L∞) of 114 mm. Our study provides the first quantitative data and histological analysis of the reproductive biology of U. tumidus, serving as a basis for future research and conservation.

Ecotoxicological evaluation and regeneration impairment of planarians by dibutyl phthalate

Commonly utilized as a plasticizer in the food and chemical sectors, Dibutyl phthalate (DBP) poses threats to the environment and human well-being as it seeps or moves into the surroundings. Nevertheless, research on the harmfulness of DBP to aquatic organisms is limited, and its impact on stem cells and tissue regeneration remains unidentified. Planarians, recognized for their robust regenerative capabilities and sensitivity to aquatic pollutants, are emerging animal models in toxicology. This study investigated the comprehensive toxicity effects of environmentally relevant levels of DBP on planarians. It revealed potential toxicity mechanisms through the use of immunofluorescence, chromatin dispersion assay, Western blot, quantitative real-time fluorescence quantitative PCR (qRT-PCR), chromatin behavioral and histological analyses, immunofluorescence, and terminal dUTP nickel-end labeling (TUNEL). Findings illustrated that DBP caused morphological and motor abnormalities, tissue damage, regenerative inhibition, and developmental neurotoxicity. Further research revealed increased apoptosis and suppressed stem cell proliferation and differentiation, disrupting a balance of cell proliferation and death, ultimately leading to morphological defects and functional abnormalities. This was attributed to oxidative stress and DNA damage caused by excessive release of reactive oxygen species (ROS). This exploration furnishes fresh perspectives on evaluating the toxicity peril posed by DBP in aquatic organisms.

FoxO3 Regulates Mouse Bone Mesenchymal Stem Cell Fate and Bone-Fat Balance During Skeletal Aging.

Age-related osteoporosis is characterized by an imbalance between osteogenic and adipogenic differentiation in bone mesenchymal stem cells (BMSCs). Forkhead box O 3 (FoxO3) transcription factor isinvolved in lifespan and cell differentiation. In this study, we explore whether FoxO3 regulates age-related bone loss and marrow fat accumulation. The expression levels of FoxO3 in BMSCs during aging were detected invivo and in vitro. To explore the role of FoxO3 in osteogenic and adipogenic differentiation, primary BMSCs were isolated from young and aged mice. FoxO3 expression was modulated by adenoviral vector transfection. The role of FoxO3 in bone-fat balance was evaluated by alizarin red S staining, oil red O staining, quantitative reverse transcription-polymerase chain reaction, Western blot, and histological analysis. Age-related bone loss and fat deposit are associated with downregulation of FoxO3. Overexpression of FoxO3 alleviated age-related bone loss and marrow fat accumulation in aged mice. Mechanistically, FoxO3 reduced adipogenesis and enhanced osteogenesis of BMSCs via downregulation of PPAR-γ and Notch signaling, respectively. In conclusion, FoxO3 is an essential factor controlling the fate of BMSCs and is a potential target for the prevention of age-related osteoporosis.

Decellularized porcine vena cava grafts are fully repopulated after orthotopic implantation

Tissue-engineered organs, based on native extracellular matrix (ECM) scaffolds, could be a game changer in regenerative medicine applications. Decellularization technology provides such scaffolds with organ-typic ECM composition and architecture. Despite limitations such as the requirement of huge cell numbers and finding the optimal route of entry, recellularized scaffolds provide alternative grafts for transplantation. In this study we assessed whether decellularized scaffolds, when implanted, are repopulated from the adjacent tissue. Since the vasculature plays an important role in tissue functionality, our main focus was to evaluate in situ repopulation of decellularized veins in a pig model. For this, porcine inferior vena cava grafts were decellularized and orthotopically implanted in recipient pigs (n = 12). To evaluate possible immune responses to the scaffolds and to assess potential thrombus formation, cellular allogeneic vena cava grafts were transplanted in control pigs (n = 8). Within 28 days after implantation, the decellularized veins were fully recellularized with endothelial cells and smooth muscle cells. Quantitative histological analysis showed a comparable amount of smooth muscle actin in the repopulated decellularized grafts similar to the native IVC. Lymphocyte infiltrates representing signs of graft rejection were not detected in the pigs, as opposed to the control group that received the allogeneic grafts. The decellularized grafts provoked a higher incidence of thrombosis in comparison with allogeneic grafts (33.3 vs. 12.5%). With this study, we show efficient in situ repopulation of decellularized vein grafts. These findings are insightful and promising to further explore the use of decellularized tissue without the need for full pre-transplant recellularization.Graphical abstract

Improved Preservation of Mouse Intestinal Tissue Using a Formalin/Acetic Acid Fixative and Quantitative Histological Analysis Using QuPath.

The architecture and morphology of the intestinal tissue from mice or other small animals are difficult to preserve for histological and molecular analysis due to the fragile nature of this tissue. The intestinal mucosa consists of villi and crypts lined with epithelial cells. In between the epithelial folds extends the lamina propria, a loose connective tissue that contains blood and lymph vessels, fibroblasts, and immune cells. Underneath the mucosa are two layers of contractile smooth muscle and nerves. The tissue experiencessignificant changes during fixation, which can impair the reliability of histologic analysis. Poor-quality histologic sections are not suitable for quantitative image-based tissue analysis. This article offers a new fixative composed of neutral buffered formalin (NBF) and acetic acid, called FA. This fixative significantly improved the histology of mouse intestinal tissue compared to traditional NBF and enabled precise, reproducible histologic molecular analyses using QuPath software. Algorithmic training of QuPath allows for automated segmentation of intestinal compartments, which can be further interrogated for cellular composition and disease-related changes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Improved preservation of mouse intestinal tissue using a formalin/acetic acid fixative Support Protocol: Quantitative tissue analysis using QuPath.

Interleukin-10 supplementation protects against oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages

Approximately 10% of the United States population will experience a kidney stone (KS). Dietary oxalate promotes calcium oxalate (CaOx) crystals, which are precursors to CaOx KS. Macrophages rely on Interleukin-10 (IL-10) anti-inflammatory cytokine signaling to phagocytose and remove CaOx crystals. We recently reported that oxalate causes macrophages to become M1-like (pro-inflammatory) as well as have increased Interleukin-6 (IL-6; pro-inflammatory) and reduced IL-10 cytokine secretion. The purpose of this study was to assess the effects of IL-10 supplementation on mouse kidneys and bone-marrow derived macrophages exposed to oxalate. We hypothesized that exogenous IL-10 treatment prevents oxalate-mediated injury in mouse kidneys and bone-marrow derived macrophages (BMDMs). Mice with a C57BL/6 background were fed Hydroxy-L-proline (HLP; an oxalate precursor) for 7 or 14 days ± IL-10 intraperitoneal injections (25 μg/kg for 2 days) before collecting kidneys and BMDMs. Real-Time Quantitative Reverse Transcription PCR, fluorescence assays, and histological analyses were performed. Mice fed HLP had increased IL-6 and M1 macrophage expression in their kidneys over time. These mice also had reduced IL-10 and M2 macrophage gene expression. In contrast, mice fed HLP with IL-10 supplementation had increased M2 macrophage levels as well as reduced inflammation in their kidneys. Consistent with these findings, BMDM isolated from mice fed HLP displayed similar results. These cells also had reduced mitochondrial membrane polarization and lysosomal staining and increased cellular ROS; whereas, exogenous IL-10 treatment protected BMDMs from oxalate mediated injury. These findings suggest that IL-10 signaling may be important for preventing oxalate-induced injury and inflammation in mouse kidneys and BMDMs. NIH R01 DK129885 (TM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sensitivity of magnetic resonance elastography to extracellular matrix and cell motility in human prostate cancer cell line-derived xenograft models

Prostate cancer (PCa) is a significant health problem in the male population of the Western world. Magnetic resonance elastography (MRE), an emerging medical imaging technique sensitive to mechanical properties of biological tissues, detects PCa based on abnormally high stiffness and viscosity values. Yet, the origin of these changes in tissue properties and how they correlate with histopathological markers and tumor aggressiveness are largely unknown, hindering the use of tumor biomechanical properties for establishing a noninvasive PCa staging system.To infer the contributions of extracellular matrix (ECM) components and cell motility, we investigated fresh tissue specimens from two PCa xenograft mouse models, PC3 and LNCaP, using magnetic resonance elastography (MRE), diffusion-weighted imaging (DWI), quantitative histology, and nuclear shape analysis. Increased tumor stiffness and impaired water diffusion were observed to be associated with collagen and elastin accumulation and decreased cell motility. Overall, LNCaP, while more representative of clinical PCa than PC3, accumulated fewer ECM components, induced less restriction of water diffusion, and exhibited increased cell motility, resulting in overall softer and less viscous properties. Taken together, our results suggest that prostate tumor stiffness increases with ECM accumulation and cell adhesion - characteristics that influence critical biological processes of cancer development.MRE paired with DWI provides a powerful set of imaging markers that can potentially predict prostate tumor development from benign masses to aggressive malignancies in patients. Statement of significanceXenograft models of human prostate tumor cell lines, allowing correlation of microstructure-sensitive biophysical imaging parameters with quantitative histological methods, can be investigated to identify hallmarks of cancer.

Preventive and therapeutic vascular photobiomodulation decreases the inflammatory markers and enhances the muscle repair process in an animal model

Photobiomodulation therapy (PBM) has shown positive effects when applied locally to modulate the inflammatory process and facilitate muscle repair. However, the available literature on the mechanisms of action of vascular photobiomodulation (VPBM), a non-invasive method of vascular irradiation, specifically in the context of local muscle repair, is limited. Thus, this study aimed to assess the impact of vascular photobiomodulation (VPBM) using a low-level laser (LLL) on the inflammatory response and the process of skeletal muscle repair whether administered prior to or following cryoinjury-induced acute muscle damage in the tibialis anterior (TA) muscles. Wistar rats (n = 85) were organized into the following experimental groups: (1) Control (n = 5); (2) Non-Injury + VPBM (n = 20); (3) Injured (n = 20); (4) Pre-VPBM + Injury (n = 20); (5) Injury + Post-VPBM (n = 20). VPBM was administered over the vein/artery at the base of the animals' tails (wavelength: 780 nm; power: 40 mW; application area: 0.04 cm2; energy density: 80 J/cm2). Euthanasia of the animals was carried out at 1, 2, 5, and 7 days after inducing the injuries. Tibialis anterior (TA) muscles were collected for both qualitative and quantitative histological analysis using H&E staining and for assessing protein expression of TNF-α, MCP-1, IL-1β, and IL-6 via ELISA. Blood samples were collected and analyzed using an automatic hematological analyzer and a leukocyte differential counter. Data were subjected to statistical analysis (ANOVA/Tukey). The results revealed that applying VPBM prior to injury led to an increase in circulating neutrophils (granulocytes) after 1 day and a subsequent increase in monocytes after 2 and 5 days, compared to the Non-Injury + VPBM and Injured groups. Notably, an increase in erythrocytes and hemoglobin concentration was observed in the Non-Injury + VPBM group on days 1 and 2 in comparison to the Injured group. In terms of histological aspects, only the Prior VPBM + Injured group exhibited a reduction in the number of inflammatory cells after 1, 5, and 7 days, along with an increase in blood vessels at 5 days. Both the Prior VPBM + Injured and Injured + VPBM after groups displayed a decrease in myonecrosis at 1, 2, and 7 days, an increase in newly-formed and immature fibers after 5 and 7 days, and neovascularization after 1, 2, and 7 days. Regarding protein expression, there was an increase in MCP-1 after 1 and 5 days, TNF-α, IL-6, and IL-1β after 1, 2, and 5 days in the Injured + VPBM after group when compared to the other experimental groups. The Prior VPBM + Injured group exhibited increased MCP-1 production after 2 days, in comparison to the Non-Injury + VPBM and Control groups. Notably, on day 7, the Injured group continued to show elevated MCP-1 protein expression when compared to the VPBM groups. In conclusion, VPBM effectively modulated hematological parameters, circulating leukocytes, the protein expression of the chemokine MCP-1, and the proinflammatory cytokines TNF-α and IL-1β, ultimately influencing the inflammatory process. This modulation resulted in a reduction of myonecrosis, restoration of tissue architecture, increased formation of newly and immature muscle fibers, and enhanced neovascularization, with more pronounced effects when VPBM was applied prior to the muscle injury.

Early treatment with rifaximin during epileptogenesis reverses gut alterations and reduces seizure duration in a mouse model of acquired epilepsy

The gut microbiota is altered in epilepsy and is emerging as a potential target for new therapies. We studied the effects of rifaximin, a gastrointestinal tract-specific antibiotic, on seizures and neuropathology and on alterations in the gut and its microbiota in a mouse model of temporal lobe epilepsy (TLE).Epilepsy was induced by intra-amygdala kainate injection causing status epilepticus (SE) in C57Bl6 adult male mice. Sham mice were injected with vehicle. Two cohorts of SE mice were fed a rifaximin-supplemented diet for 21 days, starting either at 24 h post-SE (early disease stage) or at day 51 post-SE (chronic disease stage). Corresponding groups of SE mice (one each disease stage) were fed a standard (control) diet. Cortical ECoG recording was done at each disease stage (24/7) for 21 days in all SE mice to measure the number and duration of spontaneous seizures during either rifaximin treatment or control diet. Then, epileptic mice ± rifaximin and respective sham mice were sacrificed and brain, gut and feces collected. Biospecimens were used for: (i) quantitative histological analysis of the gut structural and cellular components; (ii) markers of gut inflammation and intestinal barrier integrity by RTqPCR; (iii) 16S rRNA metagenomics analysis in feces. Hippocampal neuronal cell loss was assessed in epileptic mice killed in the early disease phase.Rifaximin administered for 21 days post-SE (early disease stage) reduced seizure duration (p < 0.01) and prevented hilar mossy cells loss in the hippocampus compared to epileptic mice fed a control diet. Epileptic mice fed a control diet showed a reduction of both villus height and villus height/crypt depth ratio (p < 0.01) and a decreased number of goblet cells (p < 0.01) in the duodenum, as well as increased macrophage (Iba1)-immunostaining in the jejunum (p < 0.05), compared to respective sham mice.Rifaximin’s effect on seizures was associated with a reversal of gut structural and cellular changes, except for goblet cells which remained reduced. Seizure duration in epileptic mice was negatively correlated with the number of mossy cells (p < 0.01) and with villus height/crypt depth ratio (p < 0.05).Rifaximin-treated epileptic mice also showed increased tight junctions (occludin and ZO-1, p < 0.01) and decreased TNF mRNA expression (p < 0.01) in the duodenum compared to epileptic mice fed a control diet.Rifaximin administered for 21 days in chronic epileptic mice (chronic disease stage) did not change the number or duration of seizures compared to epileptic mice fed a control diet. Chronic epileptic mice fed a control diet showed an increased crypt depth (p < 0.05) and reduced villus height/crypt depth ratio (p < 0.01) compared to respective sham mice. Rifaximin treatment did not affect these intestinal changes.At both disease stages, rifaximin modified α- and β-diversity in epileptic and sham mice compared to respective mice fed a control diet. The microbiota composition in epileptic mice, as well as the effects of rifaximin at the phylum, family and genus levels, depended on the stage of the disease. During the early disease phase, the abundance of specific taxa was positively correlated with seizure duration in epileptic mice.In conclusion, gut-related alterations reflecting a dysfunctional state, occur during epilepsy development in a TLE mouse model. A short-term treatment with rifaximin during the early phase of the disease, reduced seizure duration and neuropathology, and reversed some intestinal changes, strengthening the therapeutic effects of gut-based therapies in epilepsy.

Fibrin Glue Coating Limits Scar Tissue Formation around Peripheral Nerves.

Scar tissue formation presents a significant barrier to peripheral nerve recovery in clinical practice. While different experimental methods have been described, there is no clinically available gold standard for its prevention. This study aims to determine the potential of fibrin glue (FG) to limit scarring around peripheral nerves. Thirty rats were divided into three groups: glutaraldehyde-induced sciatic nerve injury treated with FG (GA + FG), sciatic nerve injury with no treatment (GA), and no sciatic nerve injury (Sham). Neural regeneration was assessed with weekly measurements of the visual static sciatic index as a parameter for sciatic nerve function across a 12-week period. After 12 weeks, qualitative and quantitative histological analysis of scar tissue formation was performed. Furthermore, histomorphometric analysis and wet muscle weight analysis were performed after the postoperative observation period. The GA + FG group showed a faster functional recovery (6 versus 9 weeks) compared to the GA group. The FG-treated group showed significantly lower perineural scar tissue formation and significantly higher fiber density, myelin thickness, axon thickness, and myelinated fiber thickness than the GA group. A significantly higher wet muscle weight ratio of the tibialis anterior muscle was found in the GA + FG group compared to the GA group. Our results suggest that applying FG to injured nerves is a promising scar tissue prevention strategy associated with improved regeneration both at the microscopic and at the functional level. Our results can serve as a platform for innovation in the field of perineural regeneration with immense clinical potential.

Combination of a human articular cartilage-derived extracellular matrix scaffold and microfracture techniques for cartilage regeneration: A proof of concept in a sheep model

BackgroundThe utilization of decellularized extracellular matrix has gained considerable attention across numerous areas in regenerative research. Of particular interest is the human articular cartilage-derived extracellular matrix (hACECM), which presents as a promising facilitator for cartilage regeneration. Concurrently, the microfracture (MF) ​technique, a well-established marrow stimulation method, has proven efficacious in the repair of cartilage defects. However, as of the current literature review, no investigations have explored the potential of a combined application of hACECM and the microfracture technique in the repair of cartilage defects within a sheep model. HypothesisThe combination of hACECM scaffold and microfracture will result in improved repair of full-thickness femoral condyle articular cartilage defects compared to the use of either technique alone. Study designControlled laboratory study. MethodsFull-thickness femoral condyle articular cartilage defect (diameter, 7.0 ​mm; debrided down to the subchondral bone plate) were created in the weight-bearing area of the femoral medial and lateral condyles (n ​= ​24). All of defected sheep were randomly divided into four groups: control, microfracture, hACECM scaffold, and hACECM scaffold ​+ ​microfracture. After 3, 6 and 12 months, the chondral repair was assessed for standardized (semi-) quantitative macroscopic, imaging, histological, immunohistochemical, mechanics, and biochemical analyses in each group. ResultAt 3, 6 and 12 months after implantation, the gross view and pathological staining of regenerative tissues were better in the hACECM scaffold and hACECM scaffold ​+ ​microfracture groups than in the microfracture and control groups; Micro-CT result showed that the parameters about the calcified layer of cartilage and subchondral bone were better in the hACECM scaffold and hACECM scaffold ​+ ​microfracture groups than the others, and excessive subchondral bone proliferation in the microfracture group. The results demonstrate that human cartilage extracellular matrix scaffold alone is an efficient, safe and simple way to repair cartilage defects. ConclusionhACECM scaffolds combined with/without microfracture facilitate chondral defect repair. The translational potential of this articlePreclinical large animal models represent an important adjunct and surrogate for studies on articular cartilage repair, while the sheep stifle joint reflects many key features of the human knee and are therefore optimal experimental model for future clinical application in human. In this study, we developed a human articular cartilage-derived extracellular matrix scaffold and to verify the viability of its use in sheep animal models. Clinical studies are warranted to further quantify the effects of hACECM scaffolds in similar settings.

The molecular response of Mytilus coruscus mantle to shell damage under acute acidified sea water revealed by iTRAQ based quantitative proteomic analysis

Mytilus coruscus is an economically important marine bivalve that lives in estuarine sea areas with seasonal coastal acidification and frequently suffers shell injury in the natural environment. However, the molecular responses and biochemical properties of Mytilus under these conditions are not fully understood. In the present study, we employed tandem mass spectrometry combined with isobaric tagging to identify differentially expressed proteins in the mantle tissue of M. coruscus under different short-term treatments, including shell-complete mussels raised in normal seawater (pH 8.1), shell-damaged mussels raised in normal seawater (pH 8.1), and acidified seawater (pH 7.4). A total of 2694 proteins were identified in the mantle, and analysis of their relative abundance from the three different treatments revealed alterations in the proteins involved in immune regulation, oxidation-reduction processes, protein folding and processing, energy provision, and cytoskeleton. The results obtained by quantitative proteomic analysis of the mantle allowed us to delineate the molecular strategies adopted by M. coruscus in the shell repair process in acidified environments, including an increase in proteins involved in oxidation-reduction processes, protein processing, and cell growth at the expense of proteins involved in immune capacity and energy metabolism. SignificanceThe impact of global ocean acidification on calcifying organisms has become a major ecological and environmental problem in the world. Mytilus coruscus is an economically important marine bivalve living in estuary sea area with seasonal coastal acidification, and frequently suffering shell injury in natural environment. Molecular responses of M coruscus under the shell damage and acute acidification is still largely unknown. For this reason, iTRAQ based quantitative proteomic and histological analysis of the mantle from M. coruscus under shell damage and acute acidification were performed, for revealing the proteomic response and possible adaptation mechanism of Mytilus under combined shell damage and acidified sea water, and understanding how the mussel mantle implement a shell-repair process under acidified sea water. Our study provides important data for understanding the shell repair process and proteomic response of Mytilus under ocean acidification, and providing insights into potential adaptation of mussels to future global change.

Antinociceptive and anti-inflammatory potential, and chemical characterization of the dichloromethane fraction of Solanum lycocarpum (Solanaceae) ripe fruits by LC-DAD-MS

Ethnopharmacological relevanceSolanum lycocarpum A. St. Hil. (Solanaceae) is a species from the Brazilian Cerrado, exhibiting several medicinal properties, being used by the population in the treatment of ulcers, bronchitis, asthma and hepatitis, which involve inflammatory processes. Aim of this studyThis study aimed to chemically characterize the dichloromethane fraction (DCM), as well as verify its antinociceptive, anti-inflammatory and antioxidant potential. Materials and methodsThe DCM fraction was obtained by partitioning the ethanol extract. The chemical constituents of the DCM fraction were characterized by LC-DAD-MS. The DPPH and FRAP assays were used to evaluate the antioxidant potential. The carrageenan-induced paw edema model was used to assess the anti-inflammatory effects, and the inflammatory infiltrate was evaluated by qualitative and quantitative histological analyses. The antinociceptive action of the DCM fraction was evaluated by acetic acid-induced abdominal writhing test, formalin-induced nociception and hot-plate test. ResultsSteroidal alkaloids solasonine, solasodine and solamargine, as well as the alkaloid peiminine/imperialine and caffeoylquinic acids, were annotated in DCM fraction by LC-DAD-MS. The DCM fraction showed antioxidative action in the in vitro DPPH and FRAP tests, as well as an anti-inflammatory effect for the three evaluated doses of 30, 100 and 300 mg/kg in the fourth and sixth hours after the administration of carrageenan. The histological analyses evidenced considerably reduction in leukocyte migration and the number of polymorphonuclear leukocytes. The study also demonstrated antinociceptive activity for the DCM fraction, which reduced abdominal writhing at three concentrations evaluated, as well as a decrease in paw licking in the formalin-induced nociception test both in the neurogenic phase and the inflammatory phase, with greater effectiveness compared to the anti-inflammatory indomethacin. The DCM fraction also increased the latency time of the animals in the hot plate test 60 min after treatment, although it did not seem to involve the opioidergic system. ConclusionThis work evidenced that the dichloromethane fraction of S. lycocarpum fruit possesses antinociceptive and anti-inflammatory potential, which supports its use in folk medicine for management inflammatory conditions.

Delamination Strength and Elastin Interlaminar Fibers Decrease with the Development of Aortic Dissection in Model Rats.

Aortic dissection (AD) is a life-threatening tear of the vascular tissue with creation of a false lumen. To explore the mechanism underlying this tissue tear, this study investigated the delamination strength of AD model rats and the histological composition of the aorta at various stages of AD development. SD rats were administrated beta-amino propionitrile for 0 (Control), 3 (Pre-dissection), and 6 (Dissection) weeks. The thoracic aorta was harvested at 10-11 weeks of age. The Dissection group exclusively showed AD at the ascending aorta. The delamination strength, a force that separates the aorta in the radial direction, of the descending aorta decreased significantly in the order of the Control, Pre-dissection, and Dissection groups. A quantitative histological analysis of the aortic tissue demonstrated that, compared with the Control group, the area fraction of collagen was significantly higher in the Pre-dissection and Dissection groups and that of elastin was significantly lower in the Dissection group. The area fraction of the elastin fibers between the elastic laminas (interlaminar fibers) was significantly decreased in the order of the Control, Pre-dissection, and Dissection groups. Histological changes of the aortic tissue, perhaps a reduction in interlaminar fibers mainly aligned in the radial direction, decreased delamination strength, thereby causing AD.

Abstract 13361: The Effect of Electronic Cigarette Exposure on Flow Mediated Vasodilation and LV Remodeling in a Chronic Myocardial Infarction Model

Introduction: There is little information about the effect of electronic cigarette (e-C) exposure on the cardiovascular system in the setting of myocardial infarction (MI) and whether e-C’s could alter vascular or cardiac function during the healing phase of MI. We determine the effect of e-C on endothelial function, cardiac remodeling and function during the healing phase of MI. Methods: Sprague Dawley rats (both sexes) were anesthetized and subjected to a proximal left coronary artery occlusion to induce a large anterior wall MI. At one week, rats were randomized to receive either 12 weeks of exposure to purified air (n=37) or e-cig vapor plus 15 mg/ml of nicotine (e-Cn=32). At 12 weeks endothelial function was tested by assessing femoral artery diameter and blood flow following a 5- minute femoral artery occlusion and reperfusion (ultrasound and flow probe). Cardiac function was assessed by echocardiography; remodeling was also assessed by quantitative histologic analysis. Results: Baseline blood flow in the femoral artery did not differ between groups, but peak reperfusion blood flow was blunted in the e-Cn group (1.59 ± 0.15 ml/min) vs. the air group (2.11 ± 0.18 ml/min; p=0.034). Femoral artery diameter after reperfusion was narrower in the e-Cn group (0.54 ± 0.02mm) compared to the air group (0.60 ± 0.02mm; p=0.023). Postmortem left ventricular (LV) volumes were similar in the e-Cn (0.69 ± 0.04 ml) and air groups (0.73 ± 0.04 ml; p=NS); and myocardial infarct expansion index did not differ between groups (1.4 ± 0.1 in e-Cn group versus 1.3 ± 0.1 in air group; p=NS). Gross pathology LV wall thickness was greater in the e-Cn group (1.38 ± 0.12 mm) versus the air group (1.04 ± 0.10 mm) despite no increase in blood pressure at 12 weeks in the e-CN group. LV fractional shortening by echo did not differ between groups at 12 weeks (e-Cn at 29 ± 2% and air at 27 ± 1%; p=NS). Conclusion: Exposure to e-Cn during the healing phase of MI was associated with altered endothelial function with reduced femoral artery blood flow and diameter at reperfusion, but not with worsened LV dilation or worsened cardiac function. E-Cn did increase gross pathologic LV wall thickness. E-Cn exposure during the healing phase of MI has deleterious effects on the CV system but does not dilate the LV.

Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells

Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

Semiautomated pipeline for quantitative analysis of heart histopathology

BackgroundHeart diseases are among the leading causes of death worldwide, many of which lead to pathological cardiomyocyte hypertrophy and capillary rarefaction in both patients and animal models, the quantification of which is both technically challenging and highly time-consuming. Here we developed a semiautomated pipeline for quantification of the size of cardiomyocytes and capillary density in cardiac histology, termed HeartJ, by generating macros in ImageJ, a broadly used, open-source, Java-based software.MethodsWe have used modified Gomori silver staining, which is easy to perform and digitize in high throughput, or Fluorescein-labeled lectin staining. The latter can be easily combined with other stainings, allowing additional quantitative analysis on the same section, e.g., the size of cardiomyocyte nuclei, capillary density, or single-cardiomyocyte protein expression. We validated the pipeline in a mouse model of cardiac hypertrophy induced by transverse aortic constriction, and in autopsy samples of patients with and without aortic stenosis.ResultsIn both animals and humans, HeartJ-based histology quantification revealed significant hypertrophy of cardiomyocytes reflecting other parameters of hypertrophy and rarefaction of microvasculature and enabling the analysis of protein expression in individual cardiomyocytes. The analysis also revealed that murine and human cardiomyocytes had similar diameters in health and extent of hypertrophy in disease confirming the translatability of our murine cardiac hypertrophy model. HeartJ enables a rapid analysis that would not be feasible by manual methods. The pipeline has little hardware requirements and is freely available.ConclusionsIn summary, our analysis pipeline can facilitate effective and objective quantitative histological analyses in preclinical and clinical heart samples.

A Quantitative Histologic Analysis of Oogenesis in the Flatfish Species Pleuronectes platessa as a Tool for Fisheries Management.

The following paper gives a detailed description of the oogenesis cycle for the European Plaice (Pleuronectes platessa), from oogonia to post-ovulatory follicle, including ovarian follicle and zona pellucida sizes. Noteworthy particularities were the difficulty in identifying cortical alveoli due to their very small size. Quantitative histology (stereology) on histological slides was used to determine a first size at maturity for females from the English Channel, which was found to be smaller compared to the literature (19 cm). Stereology also determined a first spawning event starting in January, with a peak in February and ongoing until March. Moreover, the use of stereology showed misclassifications for individuals categorized into a maturity phase using a macroscopic visual method. Misclassifications were found with individuals that had spawned (D) but were put under the immature (A) phase, and individuals in development (B) classified under D.