Proliferation and apoptosis dynamics of the normal canine mammary gland during the oestrous cycle evaluated by Ki-67 and Caspase-3 indexes.

Understanding the normal physiology of the canine mammary gland (CMG) is crucial, as it provides a foundational reference for understanding canine mammary neoplasms. The relation between the Proliferation Index (PI) indicated by Ki-67 expression, along with the Apoptotic Index (AI) determined through Caspase-3 expression during the oestrous cycle, is inadequately documented in existing literature. This study seeks to offer insights into the interplay between PI and AI in the CMG across oestrous cycle phases. An extensive investigation was conducted on a diverse case series of bitches (n = 18). Oestrous cycle stages were determined through vaginal cytology, histological examination of the reproductive tract and serum progesterone and oestradiol concentrations. The entire mammary chain was histologically examined, and proliferation and apoptosis were assessed via double immunohistochemistry employing anti-Ki-67 and Caspase-3 antibodies. PI and AI were evaluated through a systematic random sampling approach, counting a minimum of 200 cells for each cell type. There was a significantly higher PI during early dioestrus in all mammary gland components, with a greater proportion of positive cells observed in epithelial cells compared to stromal cells. The highest PI was detected in epithelial cells within the end buds. Significant differences were found in Ki-67 labelling across the cranial mammary glands. A positive and strong correlation was noted between progesterone concentration and PI in epithelial cells. The AI remained consistently low throughout the oestrous cycle, with few differences observed across histological components. Caspase-3 labelling displayed the highest positivity in caudal mammary pairs. A negative and moderate correlation was identified between progesterone concentration and AI in interlobular mesenchymal cells. This study highlights the influence of endocrine regulation on cell proliferation indices in mammary tissue, emphasizing the need to consider these hormonal variations in toxicopathological studies involving canine mammary gland.

Understanding Travel Considerations and Barriers for People with Disabilities to Using Current Modes of Transportation Through Journey Mapping.

This study aimed to apply a journey mapping methodology to identify travel considerations and barriers for people with disabilities (PWDs) at each travel stage, from considering a trip through to arriving at the destination for their current modes of transportation, with the objective of understanding and avoiding "pain points" during a transition to autonomous driving systems. Twenty PWDs, including those with physical, visual, aural, cognitive, and combined physical/visual impairments, participated in a semistructured one-on-one interview. Descriptive statistics were used for demographic information, and qualitative content analysis was used to analyze the transcribed interviews and extract themes. Themes were further organized by the modes of transportation used. The top four themes in considering and planning a trip were third-party assistance availability (private vehicle, public transportation, and paratransit), finding an accessible or suitable parking space (private vehicle), access to a service location (public transportation and paratransit), and transportation schedules (public transportation and paratransit). The top four travel barriers to locating, entering, riding, and exiting transportation and arriving at the destination were vehicle ingress/egress (private vehicle and public transportation), concerns about wheelchair securement (public transportation and paratransit), requiring third-party assistance (private vehicle and public transportation), and accessibility to service locations (public transportation). The study suggests that to mitigate travel considerations and barriers for PWDs, vehicle-specific barriers and infrastructure issues should be addressed simultaneously. We anticipate that the findings will provide insights into the design and development of autonomous vehicles, to better accommodate the needs of PWDs.

The Identified Skeleton Collection of Évora: importance for forensic science and bioarchaeology in the southern inland of Portugal.

The importance of reference osteological collections is unquestionable. However, the development of methodologies is more reliable the closer the collection is socioeconomically, demographically, and genetically to the population under study. The purpose of this study is to characterize and contextualize a new Portuguese reference collection. The collection of identified skeletons from the University of Évora comprises 201 adults of both sexes and seven non-adults, deceased between 1870 and 1993 and born between 1790 and 1969. It consists almost exclusively of individuals who were born and died in inland Alentejo, being the only Portuguese collection with this characteristic. The collection was built following all current legal and ethical obligations. Due to its characteristics, the collection constitutes a fundamental tool for forensic and bioarchaeological research in the inland regions of Portugal. In addition to the possibility offered to develop and validate methodologies in both sciences, the available complete hospital archives for research make this collection unique for health studies before, during, and after the epidemiological transition.

Mycobacterium leprae diversity and population dynamics in medieval Europe from novel ancient genomes.

BACKGROUND: Hansen's disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, Mycobacterium leprae, can significantly improve our understanding of the disease's complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period. RESULTS: Here, we reconstructed 19 ancient M. leprae genomes to further investigate M. leprae's genetic variation in Europe, with a dedicated focus on bacterial genomes from previously unstudied regions (Belarus, Iberia, Russia, Scotland), from multiple sites in a single region (Cambridgeshire, England), and from two Iberian leprosaria. Overall, our data confirm the existence of similar phylogeographic patterns across Europe, including high diversity in leprosaria. Further, we identified a new genotype in Belarus. By doubling the number of complete ancient M. leprae genomes, our results improve our knowledge of the past phylogeography of M. leprae and reveal a particularly high M. leprae diversity in European medieval leprosaria. CONCLUSIONS: Our findings allow us to detect similar patterns of strain diversity across Europe with branch 3 as the most common branch and the leprosaria as centers for high diversity. The higher resolution of our phylogeny tree also refined our understanding of the interspecies transfer between red squirrels and humans pointing to a late antique/early medieval transmission. Furthermore, with our new estimates on the past population diversity of M. leprae, we gained first insights into the disease's global history in relation to major historic events such as the Roman expansion or the beginning of the regular transatlantic long distance trade. In summary, our findings highlight how studying ancient M. leprae genomes worldwide improves our understanding of leprosy's global history and can contribute to current models of M. leprae's worldwide dissemination, including interspecies transmissions.

Assessing the association between vitamin D receptor and dental age variability.

OBJECTIVES: To explore the association between genetic polymorphisms in vitamin D receptor (VDR), vitamin D serum levels, and variability in dental age. MATERIAL AND METHODS: This cross-sectional study was based on an oral examination, panoramic radiograph analysis, and genotype analysis from biological samples. Dental age was evaluated using two different methods: Demirjian et al. (Hum Biol 45:211-227, 1973) and Hofmann et al. (J Orofac Orthop.78:97-111, 2017). The genetic polymorphisms BglI (rs739837) and FokI (rs2228570) in VDR were genotyped through real-time PCR. The vitamin D level was also measured in the serum. Delta (dental age-chronological age) was compared among genotypes in VDR in the co-dominant model. Multiple linear regression analysis was also performed. An established alpha of 5% was used. RESULTS: Genotype distributions of BglI and FokI were not associated with dental maturity (p > 0.05). In the logistic regression analyses, genotypes in BglI and FokI and vitamin D levels were not associated with variability in dental age (p > 0.05). CONCLUSIONS: The genetic polymorphisms BglI and FokI in VDR and the vitamin D levels were not associated with variability in dental age. CLINICAL RELEVANCE: To unravel the factors involved in dental maturity can improve dental treatment planning in pediatric and orthodontic practice.

Stereological assessment of sexual dimorphism in the rat liver reveals differences in hepatocytes and Kupffer cells but not hepatic stellate cells.

There is long-standing evidence that male and female rat livers differ in enzyme activity. More recently, differences in gene expression profiling have also been found to exist; however, it is still unclear whether there is morphological expression of male/female differences in the normal liver. Such differences could help to explain features seen at the pathological level, such as the greater regenerative potential generally attributed to the female liver. In this paper, hepatocytes (HEP), Kupffer cells (KC) and hepatic stellate cells (HSC) of male and female rats were examined to investigate hypothesised differences in number, volume and spatial co-localisation of these cell types. Immunohistochemistry and design-based stereology were used to estimate total numbers, numbers per gram and mean cell volumes. The position of HSC within lobules (periportal vs. centrilobular) and their spatial proximity to KC was also assessed. In addition, flow cytometry was used to investigate the liver ploidy. In the case of HEP and KC, differences in the measured cell parameters were observed between male and female specimens; however, no such differences were detected for HSC. Female samples contained a higher number of HEP per gram, with more binucleate cells. The HEP nuclei were smaller in females, which was coincident with more abundant diploid particles in these animals. The female liver also had a greater number of KC per gram, with a lower percentage of KC in the vicinity of HSC compared with males. In this study, we document hitherto unknown morphological sexual dimorphism in the rat liver, namely in HEP and KC. These differences may account for the higher regenerative potential of the female liver and lend weight to the argument for considering the rat liver as a sexually dimorphic organ.

A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. Binding to canine gastric mucosa with defined gastric glycophenotype.

BACKGROUND: The gastric mucosa of dogs is often colonized by non-Helicobacter pylori helicobacters (NHPH), while H. pylori is the predominant gastric Helicobacter species in humans. The colonization of the human gastric mucosa by H. pylori is highly dependent on the recognition of host glycan receptors. Our goal was to define the canine gastric mucosa glycophenotype and to evaluate the capacity of different gastric Helicobacter species to adhere to the canine gastric mucosa. MATERIALS AND METHODS: The glycosylation profile in body and antral compartments of the canine gastric mucosa, with focus on the expression of histo-blood group antigens was evaluated. The in vitro binding capacity of FITC-labeled H. pylori and NHPH to the canine gastric mucosa was assessed in cases representative of the canine glycosylation pattern. RESULTS: The canine gastric mucosa lacks expression of type 1 Lewis antigens and presents a broad expression of type 2 structures and A antigen, both in the surface and glandular epithelium. Regarding the canine antral mucosa, H. heilmannii s.s. presented the highest adhesion score whereas in the body region the SabA-positive H. pylori strain was the strain that adhered more. CONCLUSIONS: The canine gastric mucosa showed a glycosylation profile different from the human gastric mucosa suggesting that alternative glycan receptors may be involved in Helicobacter spp. binding. Helicobacter pylori and NHPH strains differ in their ability to adhere to canine gastric mucosa. Among the NHPH, H. heilmannii s.s. presented the highest adhesion capacity in agreement with its reported colonization of the canine stomach.

Ptaquiloside-induced early-stage urothelial lesions show increased cell proliferation and intact ß-catenin and E-cadherin expression.

Bracken (Pteridium aquilinum) is a carcinogenic plant whose main toxin, ptaquiloside, causes cancer in farm and laboratory animals. Ptaquiloside contaminates underground waters as well as meat and milk from bracken-grazing animals and is a suspected human carcinogen. A better understanding of the underlying mechanisms of carcinogenesis can be achieved by studying the early stages of this process. Unfortunately, most research on ptaquiloside has focused on the late, malignant, lesions, so the early changes of ptaquiloside-induced carcinogenesis remain largely unknown. This study aims to characterize early-stage ptaquiloside-induced urinary bladder lesions both morphologically and immunohistochemically. 12 male CD-1 mice were administered 0.5 mg ptaquiloside intraperitoneally, weekly, for 15 weeks, followed by 15 weeks without treatment. 12 control animals were administered saline. Bladders were tested immunohistochemically for antibodies against a cell proliferation marker (Ki-67), and two cell adhesion markers (E-cadherin and ß-catenin). Two exposed animals died during the work. Six ptaquiloside-exposed mice developed low-grade and two developed high grade urothelial dysplasia. No lesions were detected on control animals. Significantly, increased (p < 0.05) Ki-67 labeling indices were found on dysplastic urothelium from ptaquiloside-exposed mice, compared with controls. No differences were found concerning E-cadherin and ß-catenin expression. Early-stage ptaquiloside-induced urothelial lesions show increased cell proliferation but there is no evidence for reduced intercellular adhesiveness, though this may be a later event in tumor progression.

Comparative histological description of the intestine in platyfish (Xiphophorus maculatus) and swordtail fish (Xiphophorus helleri).

This study aimed to provide a comprehensive analysis of the histological structure of intestinal tissues of platyfish (Xiphophorus maculatus) and swordtail fish (Xiphophorus helleri). Specifically, the objectives were: (1) to compare the structural adaptations of their intestines related to their distinct feeding habits, diet, and digestive strategies; and (2) to explore their potential as animal models for intestinal disease research. Through detailed examination of tissue morphology, cell types, and structural features, this study found that both species lack a stomach, with the intestine directly connected to the esophagus. Additionally, this study proposes a new division of the intestine into anterior and posterior segments based on distinct histological characteristics. The anterior segment may be adapted for temporary food storage and digestion and was characterized by elongated epithelial cells and thin intestinal folds. In contrast, the posterior segment displayed shorter villi and higher concentrations of goblet cells. This study is the first to describe in detail the intestinal morphology of platyfish and swordtail fish. These findings contribute significantly to the understanding of the comparative anatomy and physiology of these fish species, highlighting their potential as valuable models for intestinal biology research.

Variability of lesions and lesion patterns in possible treponematosis cases: Insights from an archaeological site in Tomar, Portugal.

Objective: This study aims to describe various types of skeletal lesions and lesion patterns observed in five potential cases of treponematosis from the same archaeological site. Material and methods: The study examines five adult skeletons recovered from Santa Maria do Olival, Tomar, Portugal. Macroscopic observations were conducted on all remains, supplemented by relevant radiographic investigations. Results: The analyzed individuals exhibited diverse types of skeletal lesions and distinct patterns of lesion distribution. Radiocarbon dating of two of these skeletons locates them chronologically close to the beginning of the colonial period (15th century). Discussion: The findings indicate considerable variation in the lesions and lesion patterns associated with treponematosis, even within the same archaeological site. The radiocarbon data implies the presence of pre-Columbian treponemal disease or a rapid progression to the tertiary phase of the disease. This study represents the oldest cases of treponematosis in Portugal and the only one with more than one individual affected from the same archaeological site. Considering Portugal's prominent role in the European Age of Exploration, the study underscores the significance of pathogen exchanges between Europeans, South Americans, Africans, and Asians. Future investigations should reassess Iberian osteological collections from this time period, given the historical importance of Portugal and Spain in the intercontinental movements.

Estrogenic Responsiveness of Brown Trout Primary Hepatocyte Spheroids to Environmental Levels of 17α-Ethinylestradiol.

Three-dimensional (3D) fish hepatocyte cultures are promising alternative models for replicating in vivo data. Few studies have attempted to characterise the structure and function of fish 3D liver models and illustrate their applicability. This study aimed to further characterise a previously established spheroid model obtained from juvenile brown trout (Salmo trutta) primary hepatocytes under estrogenic stimulation. The spheroids were exposed for six days to environmentally relevant concentrations of 17α-ethinylestradiol-EE2 (1-100 ng/L). The mRNA levels of peroxisome (catalase-Cat and urate oxidase-Uox), lipid metabolism (acyl-CoA long chain synthetase 1-Acsl1, apolipoprotein AI-ApoAI, and fatty acid binding protein 1-Fabp1), and estrogen-related (estrogen receptor α-ERα, estrogen receptor ß-ERß, vitellogenin A-VtgA, zona pellucida glycoprotein 2.5-ZP2.5, and zona pellucida glycoprotein 3a.2-ZP3a.2) target genes were evaluated by quantitative real-time polymerase chain reaction. Immunohistochemistry was used to assess Vtg and ZP protein expressions. At the highest EE2 concentration, VtgA and ZP2.5 genes were significantly upregulated. The remaining target genes were not significantly altered by EE2. Vtg and ZP immunostaining was consistently increased in spheroids exposed to 50 and 100 ng/L of EE2, whereas lower EE2 levels resulted in a weaker signal. EE2 did not induce significant changes in the spheroids' viability and morphological parameters. This study identified EE2 effects at environmentally relevant doses in trout liver spheroids, indicating its usefulness as a proxy for in vivo impacts of xenoestrogens.

Influence of silver fluoride plus potassium iodine on adhesive properties of primary carious dentin-resin interface: An in vitro study.

This paper evaluated the influence of different protocols of silver fluoride (SF) pretreatment of artificial carious lesions on the adhesive interface of composite resin restorations and remineralization of deciduous dentin compared to silver diamine fluoride (SDF). Sixty-four deciduous molar teeth were randomly divided into 8 groups (n = 8) according to the restoration time (immediately - IM; 30 days after SDF/SF treatment - 30 D) and treatment before restoration (SDF 38 %; SDF 38 % + potassium iodide - KI; SF 38 %; SF 38 % +KI). After SDF/SF application, teeth in the IM group were restored with self-etch universal adhesive system/composite resin. Samples in the 30D groups were stored in artificial saliva (37 °C) for 30 days before receiving the same restoring protocol. Beams were obtained from all groups and subjected to bond strength tests (µTBS), ultrastructural qualitative analysis (FEG) and mineral analysis (SEM/EDX; Micro-Raman spectroscopy). The µTBS data were subjected to three-factor ANOVA and multiple comparisons (Holm-Sidak method). Bond strength values (MPa) for IM groups were 16.9 ± 2.7 (SDF); 17.6 ± 3.5 (SDF + KI); 16.8 ± 5.5 (SF); 18.4 ± 4.1 (SF + KI); and 14.9 ± 4.2 (SDF); 16.0 ± 5.4 (SDF + KI); 14.1 ± 3.6(SF); 16.4 ± 5.4 (SF + KI) for 30D groups. Bond strength wasn't influenced by the moment of restoration (IM or 30D); the use of KI didn't alter adhesion characteristics; SDF/SF solutions resulted in similar adhesive strength; calcium and phosphate expressions were identified at the interfaces on IM and 30D moments. However, 30D presented qualitative increase in these ions, compatible with remineralization. It was concluded that the adhesion of composite resin restorations in artificial caries lesions of deciduous teeth treated with SDF (38 %) and SF (38 %) had similar effects in vitro; the use of KI or the moment when restorations were accomplished did not influence the adhesion and all tested protocols promoted remineralization of carious dentin.

Demirjian's and Cameriere's Methods for the Assessment of Dental Age Estimation in Children from a Southern Brazilian City.

The chronological age estimation of living individuals is a crucial part of forensic practice and clinical practice, such as in orthodontic treatment. It is well-known that methods for age estimation in living children should be tested on different populations. Ethnic affiliations in Brazil are divided into several major groups depending on the region, with the south of Brazil being known for its German immigration. (1) Background: This study aimed to evaluate the correlation between chronological age and dental age using Demirjian's method and Cameriere's method in a group of children from Joinville, South Brazil to investigate if both methods can be used to estimate dental age in this population. (2) Methods: The sample consisted of 229 panoramic radiographs (119 were males and were 110 females) from Brazilian children (ages ranging from 6 to 12 years). The chronological age at the time of the panoramic radiographic exam was calculated for each child. The dental age was estimated according to Demirjian's method and Cameriere's method. All continuous data were tested for normality by using the Shapiro-Wilk test. The Pearson correlation coefficient test was applied. An alpha of 5% (p < 0.05) was used for all analyses. (3) Results: The mean chronological age was 8.75 years. According to Demirjian's method, the mean dental age was 9.3 years, while according to Cameriere's method, the mean dental age was 8.66 years. A strong correlation between chronological age and dental age according to Demirjian (r = 0.776 and p < 0.0001) and Cameriere (r = 0.735 and p < 0.0001) was observed for both genders. (4) Conclusions: Both methods presented a good correlation with chronological age in the studied population and could be used to assess dental age in this population.

Identification of prognostic factors in canine mammary malignant tumours: a multivariable survival study.

BACKGROUND: Although several histopathological and clinical features of canine mammary gland tumours have been widely studied from a prognostic standpoint, considerable variations in tumour individual biologic behaviour difficult the definition of accurate prognostic factors. It has been suggested that the malignant behaviour of tumours is the end result of several alterations in cellular physiology that culminate in tumour growth and spread. Accordingly, the aim of this study was to determine, using a multivariable model, the independent prognostic value of several immunohistochemically detected tumour-associated molecules, such as MMP-9 and uPA in stromal cells and Ki-67, TIMP-2 and VEGF in cancer cells. RESULTS: Eighty-five female dogs affected by spontaneous malignant mammary neoplasias were followed up for a 2-year post-operative period. In univariate analysis, tumour characteristics such as size, mode of growth, regional lymph node metastases, tumour cell MIB-1 LI and MMP-9 and uPA expressions in tumour-adjacent fibroblasts, were associated with both survival and disease-free intervals. Histological type and grade were related with overall survival while VEGF and TIMP-2 were not significantly associated with none of the outcome parameters. In multivariable analysis, only a MIB-1 labelling index higher than 40% and a stromal expression of MMP-9 higher than 50% retained significant relationships with poor overall and disease-free survival. CONCLUSIONS: The results of this study indicate that MMP-9 and Ki-67 are independent prognostic markers of canine malignant mammary tumours. Furthermore, the high stromal expressions of uPA and MMP-9 in aggressive tumours suggest that these molecules are potential therapeutic targets in the post-operative treatment of canine mammary cancer.

A Step Forward in the Characterization of Primary Brown Trout Hepatocytic Spheroids as Experimental Models.

Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds.

Detection of Lymphoid Markers (CD3 and PAX5) for Immunophenotyping in Dogs and Cats: Comparison of Stained Cytology Slides and Matched Cell Blocks.

Immunolabeling on Romanowsky-stained cytology (RSC) slides can be used, although there is limited evidence of its suitability for phenotyping canine and feline lymphomas. A comparison with matched cell blocks (CB) is missing. Immunolabeling on RSC and CB was compared for lymphoid markers (CD3 and PAX5) in 53 lymphomas and 4 chylous effusions from dogs and cats. The influence of pre-analytical variables (species, time of archive, type of specimens and coverslipping) and the interobserver agreement among the 2 observers was assessed. Fewer CD3+ lymphocytes were identified in RSC, while the PAX5 positivity by RSC and CB had a substantial agreement. Immunodetection of CD3 and the diagnosis of a T-cell population on RSC was more difficult. Lower intensity and higher background were noted in RSC. Immunophenotyping was inconclusive in 54% RSC and 19% CB. The interobserver reproducibility of immunophenotyping on CB was substantial, being higher than in RSC. The immunolabeling performance on the RSC of effusion and feline samples was unsatisfactory. The detection of lymphoid markers, especially membranous antigens in retrospective RSC, is affected by the pre-analytical variables: species, time of the archive, and type of specimens. CB are a more consistent type of sample for immunophenotyping purposes.

Accessible autonomous transportation and services: a focus group study.

PURPOSE: Existing automated vehicle transportation guidelines and regulations have minimal guidance to address the specific needs of people with disabilities. Accessibility should be at the forefront to increase autonomy and independence for people with disabilities. The purpose of this research is to better understand potential facilitators and barriers to using accessible autonomous transportation. METHODS: Focus groups were conducted with key stakeholders derived from people with disabilities (n = 5), travel companions/caregivers (n = 5), and transportation experts or designers (n = 11). RESULTS: The themes include describing stakeholder perceptions across all three groups by identified themes: autonomous vehicle assistive technology, autonomy vs automation, cost, infrastructure, safety & liability, design challenges, and potential impact. CONCLUSION: Specific gaps and needs were identified regarding barriers and facilitators for transportation accessibility and evidence-based guidance. These specific gaps can help to formulate design criteria for the communication between, the interior and exterior of accessible autonomous vehicles.

Accessible and affordable autonomous transportation may increase mobility and the autonomy of people with disabilities to travel spontaneously.Autonomous vehicles and services should be designed to accommodate various types of disabilities such as multimodal and multilingual device communication.Safety and liability regulatory protocols need to be developed for incidents and emergencies.Wheelchair user, especially people who use powered devices, would need systems for ingress/egress, docking, and occupant restraints.

Establishing brown trout primary hepatocyte spheroids as a new alternative experimental model-Testing the effects of 5α-dihydrotestosterone on lipid pathways.

Three-dimensional (3D) fish liver cultures mimic the in vivo cellular microenvironment, which is ideal for ecotoxicological research. Despite that, the application of these cultures to evaluate toxic effects in fish is scarce. A 3D model of brown trout (Salmo trutta f. fario) primary hepatocyte spheroids was optimized in this study by using DMEM/F-12 with 15 mM of HEPES, 10 mL/L of an antibiotic and antimycotic solution and FBS 10% (v/v), at 18 °C with ∼100 rpm. The selection of optimal conditions was based on a multiparametric characterization of the spheroids, including biometry, viability, microanatomy and immunohistochemistry. Biometric and morphologic stabilization of spheroids was reached within 12-16 days of culture. To our knowledge, this study is the first to culture and characterize viable spheroids from brown trout primary hepatocytes for over 30 days. Further, the 3D model was tested to explore the androgenic influences on lipidic target genes after 96 h exposures to control, solvent control, 10 and 100 µM of 5α-dihydrotestosterone (DHT), a non-aromatizable androgen. Spheroids exposed to 100 µM of DHT had decreased sphericity. DHT at 100 µM also significantly down-regulated Acox1-3I, PPARγ and fatty acid synthesis targets (i.e., ACC), and significantly up-regulated Fabp1. Acsl1 was significantly up-regulated after exposure to both 10 and 100 µM of DHT. The results support that DHT modulates distinct lipidic pathways in brown trout and show that this 3D model is a new valuable tool for physiological and toxicological mechanistic studies.

Doing more with less: multiple uses of a single slide in veterinary cytology. A practical approach.

Veterinary cytology faced a remarkable evolution in the last 15 years, in part due to increase recognition of the advantages of the cytology by veterinary clinicians. Simultaneously, there has been a growing awareness by the owners about the importance of a complete diagnostic workup aimed at defining a proper treatment protocol. With the extended use of cytology, challenging diagnostic cases are more frequent, and more clinically useful answers are requested. In this scenario, the use of cytology specimens to perform ancillary techniques is a valid approach. Rather than being simply archived, cytology slides can be a valuable source and a good platform to carry out cytochemistry, immunocytochemistry, and molecular techniques. Therefore, several diagnostic techniques can be applied in tiny samples, thus following the "doing more with less" principle. The aim of this approach is to refine the cytologic diagnosis and provide additional prognostic and therapeutic information. Herein, we detailed this principle in veterinary cytology and reviewed the use of cytology specimens for ancillary techniques as a single procedure, i.e., using the whole slide, or multiple procedures, i.e., multiple procedures applied in the same slide.

Deciphering influences of testosterone and dihydrotestosterone on lipid metabolism genes using brown trout primary hepatocytes.

Despite of physiological and toxicological relevance, the potential of androgens to influence fish lipid metabolism remains poorly explored. Here, brown trout primary hepatocytes were exposed to six concentrations (1 nM to 100 µM) of dihydrotestosterone (DHT) and testosterone (T), to assess changes in the mRNA levels of genes covering diverse lipid metabolic pathways. Acsl1, essential for fatty acid activation, was up-regulated by T and DHT, whereas the lipogenic enzymes FAS and ACC were up-regulated by the highest (100 µM) concentration of T and DHT, respectively. ApoA1, the major component of high-density lipoprotein (HDL), was down-regulated by both androgens. PPARγ, linked to adipogenesis and peroxisomal ß-oxidation, was down-regulated by T and DHT, while Acox1-3I, rate-limiting in peroxisomal ß-oxidation, was down-regulated by T. Fabp1, StAR and LPL were not altered. Our findings suggest that androgens may impact on lipid transport, adipogenesis and fatty acid ß-oxidation and promote lipogenesis in fish liver.