Isolation-protocol, characterization, and in-vitro performance of equine umbilical vein endothelial cells.

Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable in-vitro models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used in vitro models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs) with relatively minimal requirements, thereby enhancing accessibility for researchers. Umbilical cords obtained from five foals were used to isolate endothelial cells, followed by morphological and immunohistochemical identification. Performance of the cells in various assays commonly used in angiogenesis research was studied. Additionally, EqUVEC expression of vascular endothelial growth factor (VEGF) was assessed using ELISA. EqUVECs exhibited endothelial characteristics, forming a homogeneous monolayer with distinctive morphology. Immunohistochemical staining confirmed positive expression of key endothelial markers including von Willebrand factor (vWF), CD31, and vascular endothelial growth factor receptor-2 (VEGFR-2). Furthermore, performance assessments in in-vitro assays demonstrated the viability, proliferation, migration, tube formation and VEGF-expression capabilities of EqUVECs. The findings suggest that EqUVECs are a promising in-vitro model for studying equine angiogenesis, offering a foundation for further investigations into equine-specific vascular processes and therapeutic interventions.

Pathohistological Findings after Bilateral Ovariectomy in Mares with Behavioral Problems.

Behavioral problems in reproductively healthy mares are a challenging issue that is successfully treated with bilateral ovariectomy (BO). This laparoscopic procedure represents an alternative to conservative treatment for mares not intended for breeding and results in high owner satisfaction regarding behavioral improvement. However, a pathohistological explanation to justify surgical ovarian removal regarding animal welfare is lacking. Therefore, the objective of this study was to pathohistologically evaluate bilaterally removed, clinically unremarkable ovaries of mares with behavioral problems (bOE, n = 20) and to compare them with pathohistologically confirmed granulosa cell tumors of mares with neoplastic ovaries (GCT-uOE, n = 10). A complete data set including preliminary presentation, clinical examination, and serum anti-Müllerian hormone (AMH) and testosterone was further analyzed in both groups. Both hormones were significantly higher in GCT-uOE compared with bOE. Immunohistochemical expression of Ki-67, AMH, aromatase, epidermal growth factor receptor, calretinin, and epithelial cadherin in granulosa cells of large follicular structures in bOE did not differ from neoplastic granulosa cells in GCT-uOE. Ultrasonographically nondetectable early neoplastic changes were pathohistologically evaluated in 15% of mares and anovulatory-like follicles in 30% of mares in bOE and might be one explanation for the high success rate of BO in 85% of bOE in this study.

Feline eosinophilic sclerosing fibroplasia associated with T-/natural killer-cell lymphoma.

Feline eosinophilic sclerosing fibroplasia (FESF) is a proliferative, inflammatory disease of the gastrointestinal tract and other sites, uncommonly diagnosed in the cat. This entity of uncertain etiology typically presents as a progressive mass lesion, mimicking a neoplastic process. In this case series, we present 17 cases of FESF associated with intralesional lymphoma. Histologic and immunohistochemical characterization of this unique lymphoma revealed that the neoplastic lymphocytes were immunopositive for CD56 and/ or CD3, suggesting a natural killer cell, natural killer T-cell, or T-cell origin. This case series represents the first description of this lymphoma subtype, for which the term eosinophilic sclerosing lymphoma is proposed.

Expression of somatostatin receptors in canine and feline meningioma.

OBJECTIVES: The standard treatment for canine and feline meningiomas includes radiotherapy, surgical excision or combined therapy. However, new therapeutic approaches are required due to the possible recurrence or progression of meningiomas despite initial therapy. Adjunctive therapy with synthetic long-acting somatostatin (SST) analogues has been described in humans with SST-expressing tumours. The expression of SST receptors (SSTRs) by feline meningiomas is currently unknown, and there are little data about canine meningiomas. We hypothesized that SSTR is expressed by canine and feline meningiomas (S1). METHODS: Seven canines and 11 felines with histologically confirmed meningiomas underwent STTR screening. RNA expressions of SSTR1, SSTR2, SSTR3 and SSTR5 (canine) and SSTR1-SSTR 5 (feline) in fresh frozen and formalin-fixed and paraffin-embedded (FFPE) samples were investigated using real-time (RT)-qPCR. The expression of SSTR1 and SSTR2 in FFPE samples was evaluated using immunohistochemistry (IHC). The specificity of applied antibodies for canine and feline species was confirmed by western blotting. RESULTS: In canine meningiomas (n = 7), RNA expression of SSTR1, SSTR2 and SSTR5 was detected in all samples; SSTR3 RNA expression was detected in only 33% of samples. In feline meningiomas (n = 12), RNA expression of SSTR1, SSTR4, SSTR5 and SSTR2 was detected in 91%, 46%, 46% and 36% of samples, respectively; SSTR3 was not expressed. Overall, the detection rate was lower in FFPE samples. IHC revealed the expression of SSTR1 and SSTR2 in all samples from both species. However, it is important to exercise caution when interpreting IHC results due to the presence of diffuse background staining. CONCLUSIONS: SSTRs are widely expressed in canine and feline meningiomas, thereby encouraging further studies investigating SSTR expression to conduct trials about the effect of adjunctive therapy with long-acting SST-analogues.

Immunophenotype investigation in feline intestinal non-B-cell lymphoma.

Lymphoma is the most common tumour of domestic cats, developing most frequently in the small intestine. Feline small intestinal lymphoma predominantly demonstrates a T-cell immunophenotype identified by standard immunopositivity for T cells with CD3 or immunopositivity for B cells with CD20. In contrast, a wide spectrum of immunohistochemical antibodies are applied in humans to diagnose the various specific lymphoma subtypes according to the WHO classification. Our aim was to augment our knowledge of immunophenotypes in feline non-B-cell lymphomas forming macroscopic masses in the intestinal tract. We evaluated the combined immunohistochemistry and flow cytometry findings from 15 cases. Neoplastic lymphoid cells were immunopositive for CD3 in 93% (14/15), granzyme B in 87% (13/15), CD5 in 20% (3/15), CD8 in 13% (2/15), CD4 in 7% (1/15) and CD56 in 7% (1/15) of cases. Cytotoxic granules indicating a cytotoxic origin of the neoplastic cells were identified by histopathology only in 13% (2/15) and by cytology in 47% (7/15) of the cases. Without immunohistochemical labelling of the cytotoxic protein granzyme B, the cytotoxic status would have been missed in 46% (6/13) of the cytological and in 85% (11/13) of the histopathological slides. These findings suggest that more complex immunophenotyping may advance our understanding and help prognosticate small intestinal T-cell lymphoma in cats.

Involvement of somatotrophic hormones in the postpartum regulation of ovarian activity in mares.

Mares resume ovarian activity rapidly after foaling. Besides follicle-stimulating hormone (FSH) and luteinizing hormone (LH), the pituitary synthesizes prolactin and growth hormone which stimulate insulin-like growth factor (IGF) synthesis in the liver. We tested the hypothesis that follicular growth is initiated already antepartum, mares with early and delayed ovulation differ in IGF-1 release and that there is an additional IGF-1 synthesis in the placenta. Plasma concentrations of LH, FSH, IGF-1, IGF-2, activin and prolactin. IGF-1, IGF-2, prolactin and their receptors in placental tissues were analyzed at the mRNA and protein level. Follicular growth was determined from 15 days before to 15 days after foaling in 14 pregnancies. Mares ovulating within 15 days postpartum formed group OV (n=5) and mares not ovulating within 15 days group NOV (n=9). Before foaling, follicles with a diameter >1 cm were present in all mares and their number increased over time (p<0.05). Follicle growth after foaling was more pronounced in OV mares (day p<0.001, group p<0.05, day x group p<0.05) in parallel to an increase in LH concentration (p<0.001, day x group p<0.001) while FSH increased (p<0.001) similarly in both groups. Plasma concentrations of IGF-1 and prolactin peaked one day after foaling (p<0.001). The IGF-1 mRNA abundance was higher in the allantochorion but lower in the amnion of OV versus NOV mares (group p=0.01, localization x group p<0.01). The IGF-1 receptor mRNA was most abundant in the allantochorion (p<0.001) and IGF-1 protein was expressed in placental tissue without differences between groups. In conclusion, follicular growth in mares is initiated before foaling and placental IGF-1 may enhance resumption of ovulatory cycles.

Endocrine changes induced by GnRH immunisation and subsequent early re-stimulation of testicular function with a GnRH agonist in stallions.

CONTEXT: Resumption of testicular function after gonadotrophin-releasing hormone (GnRH) immunisation varies among individual animals and some stallions regain fertility only after a prolonged time. AIMS: This study evaluated endocrine effects of GnRH immunisation and early subsequent re-stimulation with a GnRH agonist. We hypothesised that GnRH agonist treatment advances resumption of normal endocrine function in GnRH-vaccinated stallions. METHODS: Shetland stallions were assigned to an experimental and a control group (n =6 each). Experimental stallions were GnRH-immunised twice, 4weeks apart. Each experimental stallion was hemicastrated together with an age-matched control animal when testosterone concentration decreased below 0.3ng/mL. Three weeks later, daily treatment with the GnRH agonist buserelin was initiated (4µg/day for 4weeks followed by 8µg/day). The remaining testicle was removed when testosterone concentration exceeded 0.5ng/mL in vaccinated stallions. Blood was collected for LH, FSH, oestradiol and anti-müllerian hormone (AMH) analyses, and testicular and epididymal tissue were conserved for real-time qPCR and histology. KEY RESULTS: GnRH vaccination reduced blood concentrations of LH and FSH, with a structural deterioration of testicular tissue and disruption of spermatogenesis. Daily buserelin treatment for approximately 60days partially restored gonadotropin secretion and induced a recovery of the functional organisation of the testicular tissue with effective spermatogenesis. CONCLUSIONS: Endocrine testicular function can be restored in GnRH-vaccinated stallions by daily low-dose buserelin treatment. The buserelin treatment protocol may potentially be improved regarding the dose, interval and duration. IMPLICATIONS: Daily buserelin treatment can be recommended for treatment of GnRH-vaccinated stallions with prolonged inhibition of testicular function.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

Feline SCCs of the Head and Neck Display Partial Epithelial-Mesenchymal Transition and Harbor Stem Cell-like Cancer Cells.

Squamous cell carcinoma of the head and neck (HNSCC) is a malignant cancer disease in humans and animals. There is ample evidence that the high plasticity of cancer cells, i.e., their ability to switch from an epithelial to a mesenchymal, endothelial, and stem cell-like phenotype, chiefly contributes to progression, metastasis, and multidrug resistance of human HNSCCs. In feline HNSCC, the field of cancer cell plasticity is still unexplored. In this study, fourteen feline HNSCCs with a known feline papillomavirus (FPV) infection status were subjected to histopathological grading and subsequent screening for expression of epithelial, mesenchymal, and stem cell markers by immunohistochemistry (IHC) and immunofluorescence staining (IF). Irrespective of the FPV infection status, all tumors except one corresponded to high-grade, invasive lesions and concurrently expressed epithelial (keratins, E-cadherin, ß-catenin) and mesenchymal (vimentin, N-cadherin, CD146) proteins. This finding is indicative for partial epithelial-mesenchymal transition (pEMT) events in the lesions, as similarly described for human HNSCCs. IF double staining revealed the presence of CD44/CD271 double-positive cells notably within the tumors' invasive fronts that likely correspond to cancer stem cells. Taken together, the obtained findings suggest that feline HNSCCs closely resemble their human counterparts with respect to tumor cell plasticity.

Abundance of Anti-Muellerian hormone in cat ovaries and correlation of its plasma concentration with animal age, weight and stage of the estrous cycle.

In female animals of different species, Anti-Müllerian hormone (AMH) is produced by follicular granulosa cells and has been associated with the ovarian follicle pool. Because concentration of AMH in plasma of ovary-intact female cats is apparently more variable than previously assumed, we have analysed AMH concentration in blood of cats (n = 93) presented for routine ovariectomy and assessed ovarian histology and AMH protein expression in the surgically removed ovaries. We hypothesised that AMH is synthesized only in preantral and small antral follicles and that plasma AMH concentration reflects the antral follicle count (AFC). Corpora lutea were detected in 35% of the female cats, whereas plasma progesterone concentration was ≥1 ng/mL in 57% of the cats. Follicular cysts were present in 15 cats (16%). Positive immunostaining for AMH protein was detected in close to all primordial and antral follicles, ovarian cysts, 70% of corpora lutea and 28% of atretic follicles. Concentration of AMH in plasma averaged 6.8 ± 0.5 ng/mL (range 1.3-21.7 ng/mL). The AFC increased with increasing AMH concentration with a moderate positive correlation between AFC and AMH (r = 0.286, p < 0.01). Plasma AMH concentration was not affected by season or cats' age, weight, stage of the estrous cycle and presence of follicular cysts. In conclusion, AMH protein is expressed in all endocrine structures of the cat ovary. While AMH is a marker for the presence of ovarian tissue, its usefulness to assess ovarian function in individual female cats is of limited value.

Immunohistochemical markers for equine granulosa cell tumors: a pilot study.

Sex cord-stromal tumors (SCSTs), generally referred to as granulosa cell tumors (GCTs) or granulosa-theca cell tumors (GTCTs) in equids, show complex compositions and variable numbers of hormone-producing cells. These tumors can be difficult to diagnose, especially in early stages. Therefore, we tested a panel of antibodies for vimentin, smooth muscle actin, laminin, Ki-67, E-cadherin, calretinin, moesin, p-ezrin, AMH, and aromatase, markers used for tumor composition and classification, progression, and prognosis in human SCSTs, on an exemplary grapefruit-size equine GCT within the left ovary of a 13-year-old mare with stallion-like behavior and elevated testosterone levels in comparison with normal ovarian tissue. The tumor showed a low proliferation rate and prominent moesin and p-ezrin staining in granulosa cells. E-cadherin, calretinin, aromatase, and AMH are suggested to be potential markers for different cell components of equine SCSTs that can support tumor diagnosis and classification.

Biodegradable, Self-Reinforcing Vascular Grafts for In Situ Tissue Engineering Approaches.

Clinically available small-diameter synthetic vascular grafts (SDVGs) have unsatisfactory patency rates due to impaired graft healing. Therefore, autologous implants are still the gold standard for small vessel replacement. Bioresorbable SDVGs may be an alternative, but many polymers have inadequate biomechanical properties that lead to graft failure. To overcome these limitations, a new biodegradable SDVG is developed to ensure safe use until adequate new tissue is formed. SDVGs are electrospun using a polymer blend composed of thermoplastic polyurethane (TPU) and a new self-reinforcing TP(U-urea) (TPUU). Biocompatibility is tested in vitro by cell seeding and hemocompatibility tests. In vivo performance is evaluated in rats over a period for up to six months. Autologous rat aortic implants serve as a control group. Scanning electron microscopy, micro-computed tomography (µCT), histology, and gene expression analyses are applied. TPU/TPUU grafts show significant improvement of biomechanical properties after water incubation and exhibit excellent cyto- and hemocompatibility. All grafts remain patent, and biomechanical properties are sufficient despite wall thinning. No inflammation, aneurysms, intimal hyperplasia, or thrombus formation are observed. Evaluation of graft healing shows similar gene expression profiles of TPU/TPUU and autologous conduits. These new biodegradable, self-reinforcing SDVGs may be promising candidates for clinical use in the future.

Isolation and Characterization of Novel Canine Osteosarcoma Cell Lines from Chemotherapy-Naïve Patients.

The present study aimed to establish novel canine osteosarcoma cell lines (COS3600, COS3600B, COS4074) and characterize the recently described COS4288 cells. The established D-17 cell line served as a reference. Analyzed cell lines differed notably in their biological characteristics. Calculated doubling times were between 22 h for COS3600B and 426 h for COS4074 cells. COS3600B and COS4288 cells produced visible colonies after anchorage-independent growth in soft agar. COS4288 cells were identified as cells with the highest migratory capacity. All cells displayed the ability to invade through an artificial basement membrane matrix. Immunohistochemical analyses revealed the mesenchymal origin of all COS cell lines as well as positive staining for the osteosarcoma-relevant proteins alkaline phosphatase and karyopherin α2. Expression of p53 was confirmed in all tested cell lines. Gene expression analyses of selected genes linked to cellular immune checkpoints (CD270, CD274, CD276), kinase activity (MET, ERBB2), and metastatic potential (MMP-2, MMP-9) as well as selected long non-coding RNA (MALAT1) and microRNAs (miR-9, miR-34a, miR-93) are provided. All tested cell lines were able to grow as multicellular spheroids. In all spheroids except COS4288, calcium deposition was detected by von Kossa staining. We believe that these new cell lines serve as useful biological models for future studies.

Ketogenic diets composed of long-chain and medium-chain fatty acids induce cardiac fibrosis in mice.

PURPOSE: Heart diseases are the leading cause of death worldwide. Metabolic interventions via ketogenic diets (KDs) have been used for decades to treat epilepsy, and more recently, also diabetes and obesity, as common comorbidities of heart diseases. However, recent reports linked KDs, based on long-chain triglycerides (LCTs), to cardiac fibrosis and a reduction of heart function in rodents. As intervention using medium-chain triglycerides (MCTs) was recently shown to be beneficial in murine cardiac reperfusion injury, the question arises as to what extent the fatty acid (FA)-composition in a KD alters molecular markers of FA-oxidation (FAO) and modulates cardiac fibrotic outcome. METHODS: The effects of LCT-KD as well as an LCT/MCT mix (8:1 ketogenic ratio) on cardiac tissue integrity and the plasma metabolome were assessed in adult male C57/BL6NRJ mice after eight weeks on the respective diet. RESULTS: Both KDs resulted in increased amount of collagen fibers and cardiac tissue was immunologically indistinguishable between groups. MCT supplementation resulted in i) profound changes in plasma metabolome, ii) reduced hydroxymethylglutaryl-CoA synthase upregulation, and mitofusin 2 downregulation, iii) abrogation of LCT-induced mitochondrial enlargement, and iv) enhanced FAO profile. Contrary to literature, mitochondrial biogenesis was unaffected by KDs. We propose that the observed tissue remodeling is caused by the accumulation of 4-hydroxy-2-nonenal protein adducts, despite an inconspicuous nuclear factor (erythroid-derived 2)-like 2 pathway. CONCLUSION: We conclude that regardless of the generally favorable effects of MCTs, they cannot inhibit 4-hydroxy-2-nonenal adduct formation and fibrotic tissue formation in this setting. Furthermore, we support the burgeoning concern about the effect of KDs on the cardiac safety profile.

Osteopontin and Ki-67 expression in World Health Organization graded canine meningioma.

Osteopontin (OPN) is a matrix protein involved in tumour initiation and progression. In human meningioma, OPN has been correlated with World Health Organization (WHO) grade, brain invasion and recurrence. The aim of this study was to investigate OPN as a possible malignancy marker in canine meningioma by correlating its expression to WHO grade and proliferative activity as measured by the Ki-67 labelling index (LI). Thirty-five formalin-fixed, paraffin-embedded canine meningioma samples were classified according to the current human WHO classification. Evaluation of OPN expression was performed by immunohistochemical (IHC) labelling and calculation of the OPN intensity score (IS), OPN IHC score and Allred score. The scores were compared with WHO grades, Ki-67 LI, location and invasiveness. Nineteen meningiomas were graded as WHO grade I (54.3%), nine as grade II (25.7%) and seven as grade III (20.0%). Twenty-six tumours were located intracranially, four were retrobulbar and five were spinal meningiomas. In all specimens OPN expression was detected in moderate to high degrees. Neither the OPN scores nor the Ki-67 LIs were correlated with WHO grades. However, the OPN IS and OPN IHC score were significantly higher in WHO grade I samples compared with grade II samples (P <0.05). The OPN IS and OPN IHC score were significantly lower in meningioma samples that invaded surrounding tissues (P = 0.01 and 0.019, respectively). The results indicate a generally high expression of OPN in canine meningioma independent of WHO grade. Further research into the role of OPN as a possible therapeutic target or predictor of recurrence is warranted.

Low plasma progesterone concentration during the early luteal phase delays endometrial development and the beginning of placentation in mares.

While detrimental effects of reduced plasma progesterone concentration in the early luteal phase on conceptus development in horses have recently been demonstrated, there is no information on associated effects on the endometrium, allantochorion (AC), and chorionic girdle (CG) in this species. We hypothesised that reduced early postovulatory progesterone concentration in pregnant horses is detrimental to endometrial function and development of the embryonic membranes and is an underlying cause of delayed conceptus development. After insemination and ovulation, mares (n = 11) were assigned to treatment (TREAT) or control (CON) during two pregnancies. In TREAT pregnancies, mares received a PGF2α analogue for four consecutive days starting on the day of ovulation with the aim to reduce progesterone secretion. Mares were left untreated in CON pregnancies and thus served as their own controls. Endometrial biopsies for analysis of histomorphology, epidermal growth factor (EGF) and EGF receptor (EGFR) mRNA and protein expression in the endometrium, AC, and CG as well as abundance of regulatory T lymphocytes (Tregs) were collected on day 34 of pregnancy. Histomorphometric analysis revealed a higher luminal endometrium and a higher CG epithelium in CON compared to TREAT pregnancies. Abundance of mRNA for EGF and EGFR was large in the endometrium, AC and CG but did not differ between TREAT and CON pregnancies. The number of endometrial regulatory T lymphocytes was reduced in TREAT compared to CON pregnancies, adding further aspects to the potentially detrimental effects of reduced progesterone concentrations on equine pregnancy.

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane.

Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

Lipid droplets and perilipins in canine osteosarcoma. Investigations on tumor tissue, 2D and 3D cell culture models.

Lipid droplets were identified as important players in biological processes of various tumor types. With emphasis on lipid droplet-coating proteins (perilipins, PLINs), this study intended to shed light on the presence and formation of lipid droplets in canine osteosarcoma. For this purpose, canine osteosarcoma tissue samples (n = 11) were analyzed via immunohistochemistry and electron microscopy for lipid droplets and lipid droplet-coating proteins (PLINs). Additionally, we used the canine osteosarcoma cell lines D-17 and COS4288 in 2D monolayer and 3D spheroid (cultivated for 7, 14, and 21 days) in vitro models, and further analyzed the samples by means of histochemistry, immunofluorescence, molecular biological techniques (RT-qPCR, Western Blot) and electron microscopical imaging. Lipid droplets, PLIN2, and PLIN3 were detected in osteosarcoma tissue samples as well as in 2D and 3D cultivated D-17 and COS4288 cells. In spheroids, specific distribution patterns of lipid droplets and perilipins were identified, taking into consideration cell line specific zonal apportionment. Upon external lipid supplementation (oleic acid), a rise of lipid droplet amount accompanied with an increase of PLIN2 expression was observed. Detailed electron microscopical analyzes revealed that lipid droplet sizes in tumor tissue were comparable to that of 3D spheroid models. Moreover, the biggest lipid droplets were found in the central zone of the spheroids at all sampling time-points, reaching their maximum size at 21 days. Thus, the 3D spheroids can be considered as a relevant in vitro model for further studies focusing on lipid droplets biology and function in osteosarcoma.

An optimized workflow for microCT imaging of formalin-fixed and paraffin-embedded (FFPE) early equine embryos.

Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate embryos was mostly done on embryos that have been stained with an X-ray dense contrast agent. Here, we describe an alternative imaging procedure that allows to visualize embryo morphology and organ development in unstained FFPE embryos. Two aspects are critical for high-quality data acquisition: (i) a proper sample mounting leaving as little as possible paraffin around the sample and (ii) an image filtering pipeline that improves signal-to-noise ratio in these inherently low-contrast data sets. The presented workflow allows overview imaging of the whole embryo proper and can be used for determination of organ volumes and development. Furthermore, we show that high-resolution interior tomographies can provide virtual histology information from selected regions of interest. In addition, we demonstrate that microCT scanned embryos remain intact during the scanning procedure allowing for a subsequent investigation by routine histology and/or immunohistochemistry. This makes the presented workflow applicable also to archival paraffin-embedded material.

Lipid droplet dynamics in healthy and pyometra-affected canine endometrium.

BACKGROUND: Accumulation of lipid droplets (LDs) was recently observed in pyometra-affected uteri. As data about their nature and function are missing we intended to compare the localization, quality and quantity of LDs in canine healthy and pyometra-affected tissues and in an in vitro model. METHODS AND RESULTS: We characterized LDs in healthy and pyometra uterine tissue samples as well as in canine endometrial epithelial cells (CEECs) in vitro by means of histochemistry, immunohistochemistry, transmission electron microscopy, western blot, and RT-qPCR. Oil Red O (ORO) staining and quantification as well as p-phenylenediamine staining showed a higher number of LDs in epithelial cells of pyometra samples. Immunohistochemistry revealed that the amount of LDs coated by perilipin2 (PLIN2) protein was also higher in pyometra samples. Transmission electron microscopy showed an increase of LD size in surface and glandular epithelial cells of pyometra samples. In cell culture experiments with CEECs, supplementation with oleic acid alone or in combination with cholesterol lead to an increased LD accumulation. The expression of PLIN2 at protein and mRNA level was also higher upon oleic acid supplementation. Most LDs were double positive for ORO and PLIN2. However, ORO positive LDs lacking PLIN2 coating or LDs positive for PLIN2 but containing a lipid class not detectable by ORO staining were identified. CONCLUSIONS: We found differences in the healthy and pyometra-affected endometrium with respect to LDs size. Moreover, several kinds of LDs seem to be present in the canine endometrium. In vitro studies with CEECs could show their responsiveness to external lipids. Since epithelial cells reacted only to oleic acid stimulation, we assume that the cyclic lipid accumulation in the canine endometrium is based mainly on triglycerides and might serve as energy provision for the developing early embryo. Further studies are necessary to verify the complex role of lipids in the healthy and pyometra-affected canine endometrium.