Dynamics of blood circulation during diving in the bottlenose dolphin (Tursiops truncatus): the role of the retia mirabilia

The retia mirabilia are vascular nets composed of small vessels dispersed among numerous veins, allowing blood storage, regulation of flow and pressure damping effects. Here, we investigated their potential role during the diving phase of the bottlenose dolphin (Tursiops truncatus). To this effect, the whole vertebral retia mirabilia of a series of dolphins were removed during post-mortem analysis and examined to assess vessel diameters, and estimate vascular volume and flow rate. We formulated a new hemodynamic model to help clarify vascular dynamics throughout the diving phase, based on the total blood volume of a bottlenose dolphin, and using data available about the perfusion of the main organs and body systems. We computed the minimum blood perfusion necessary to the internal organs, and the stroke volume and cardiac output during the surface state. We then simulated breath-holding conditions and perfusion of the internal organs under the diving-induced bradycardia and reduction of stroke volume and cardiac output, using 10 beats min 121 as the limit for the heart rate for an extended dive of over 3 min.Within these simulated conditions, the retia mirabilia play a vital role as reservoirs of oxygenated blood that permit functional performances and survival of the heart and brain. Our theoretical model, based on the actual blood capacity of the retia mirabilia and available data on organ perfusion, considers the dynamic trend of vasoconstriction during the diving phase and may represent a baseline for future studies on the diving physiology of dolphins and especially for the blood supply to their brain

Array Comparative Genomic Hybridization Analysis Reveals Significantly Enriched Pathways in Canine Oral Melanoma

Human Mucosal Melanoma (hMM) is an aggressive neoplasm of neuroectodermal origin with distinctive features from the more common cutaneous form of malignant melanoma (cMM). At the molecular level, hMMs are characterized by large chromosomal aberrations rather than single-nucleotide mutations, typically observed in cMM. Given the scarcity of available cases, there have been many attempts to establish a reliable animal model. In pet dogs, Canine Oral Melanoma (COM) is the most common malignant tumor of the oral cavity, sharing clinical and histological aspects with hMM. To improve the knowledge about COM\u2019s genomic DNA alterations, in the present work, formalin-fixed, paraffin-embedded (FFPE) samples of COM from different European archives were collected to set up an array Comparative Genomic Hybridization (aCGH) analysis to estimate recurrent Copy Number Aberrations (CNAs). DNA was extracted in parallel from tumor and healthy fractions and 19 specimens were successfully submitted to labeling and competitive hybridization. Data were statistically analyzed through GISTIC2.0 and a pathway-enrichment analysis was performed with ClueGO. Recurrent gained regions were detected, affecting chromosomes CFA 10, 13 and 30, while lost regions involved chromosomes CFA 10, 11, 22, and 30. In particular, CFA 13 showed a whole-chromosome gain in 37% of the samples, while CFA 22 showed a whole-chromosome loss in 25%. A distinctive sigmoidal trend was observed in CFA 10 and 30 in 25 and 30% of the samples, respectively. Comparative analysis revealed that COM and hMM share common chromosomal changes in 32 regions. MAPK- and PI3K-related genes were the most frequently involved, while pathway analysis revealed statistically significant perturbation of cancer-related biological processes such as immune response, drug metabolism, melanocytes homeostasis, and neo-angiogenesis. The latter is a new evidence of a significant involvement of neovascularization-related pathways in COMs and can provide the rationale for future application in anti-cancer targeted therapies

Cytologic scoring of equine exercise-induced pulmonary hemorrhage: Performance of human experts and a deep learning-based algorithm

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis

Cutaneous angiomatosis-like presentation in koi carp (Cyprinus carpio koi): Clinical–pathological investigations

The skin represents an indicator of an animal's health status. Causes of cutaneous diseases in fish most often trace back to biological agents. However, fish skin diseases can also arise from a complex interaction of infectious and non-infectious causes, making it more difficult to identify a specific aetiology. In the period between April and September of the years 2019–2022, four koi carp (Cyprinus carpio koi) from two European countries presented with multifocal, irregularly round, few mm to 1 cm, variably raised cutaneous reddened areas. The fish displayed good general condition. Cutaneous samples, investigated by microbiological and molecular methods and microscopy, did not indicate a primary pathogenic agent. Gross and histological findings of the cutaneous biopsies were consistent with a multifocal/reactive process centred on dermal vessels. The histological features were reminiscent of angiomatosis, a benign proliferative condition affecting the dermal vessels of mammals, including human patients. The clinical–pathological presentation and the dermatologic condition that affected the koi carp are discussed and compared with the veterinary and human literature

Cytologic scoring of equine exercise-induced pulmonary hemorrhage : performance of human experts and a deep learning-based algorithm

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator’s and algorithmic performance included a ground truth dataset, the mean annotators’ THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.The Dres. Jutta and Georg Bruns-Stifung für innovative Veterinärmedizin.https://journals.sagepub.com/home/vetCompanion Animal Clinical Studie

Human cancers challenge a pet model: the case of Canine Oral Melanoma

Human Mucosal Melanoma (hMM) is a rare aggressive neoplasm of neuroectodermal origin characterized by large chromosomal aberrations and a low burden of single-nucleotide polymorphisms (SNPs). Given the scarcity of available cases, pet dogs have been proposed as a reliable animal model. In pet dogs, Canine Oral Melanoma (COM) is the most common malignant tumor of the oral cavity, sharing clinical, histological, and genomic characteristics with hMM. However, in the last few years, together with the deepening of the genome-wide characterization of COMs, doubts raised from some authors on the reliability of the canine spontaneous model. To clarify these points, we thoroughly reviewed the recent literature and performed our analyses on both single genes and the whole genome. The goal was to overcome the considerations limited to the classic gene comparison and discover the common biologic properties that are altered in melanoma cancer cells, to reveal the complex path of events that ensures the survival and growth of the neoplastic population. The genome-wide analysis of the cohort revealed a profile of gained and lost chromosomal regions confirming most of the results available in the literature, while no consistent point mutations were detected in the target genes analyzed. COM, therefore, confirms to be a simpler neoplastic entity than hMM when considering the mutational burden, being instead a suitable model for the complex aberration profile, which activates or inhibits biological processes related to cancer survival and proliferation. Statistically significant dysregulated pathways, such as cellular proliferation, telomere maintenance, Tyrosine-, MAP-, PI3- kinases, and melanocytes homeostasis, are shared between COM and hMM. These similarities, however, are visible only when comparison is not limited to single aberrated driver genes and robust software-based analysis is performed. Furthermore, new evidence suggests that angiogenetic pathways could be of great relevance to the advancement of targeted therapies, which need further exploration in the next future. Altogether, the results of the scientific community confirm the closeness of genomic aberrations and pathways activation of COM and hMM. Further evidences could come from the developing epigenetic analyses and the in vitro and in vivo trial of new targeted therapies

THE APPLICATION OF ARRAY COMPARATIVE GENOMIC HYBRIDIZATION IN ARCHIVAL CANINE ORAL MELANOMA REVEALS SIGNIFICANTLY ENRICHED PATHWAYS

Introduction: Human mucosal melanoma (HMM), a distinct biological entity from cutaneous melanoma, frequently harbours chromosomal rearrangement instead of somatic mutations, and is an aggressive neoplasm with severe prognosis in both man and dogs. Canine oral melanoma (COM) is currently considered a powerful model for HMM. To investigate chromosomal copy number aberrations (CNAs) in COM, array comparative genomic hybridization (aCGH) was applied to archival tissue material. Materials and Methods: Forty formalin-fixed and paraffin wax-embedded samples of treatment-na\uefve COM were selected from three European archives, with the presence of healthy tissue in the specimen a minimum inclusion criteria. DNA was extracted in parallel from tumour and healthy fractions and 19 specimens were subjected successfully to labelling and competitive hybridization. Data were analysed statistically through GISTIC2.0 and a pathway-enrichment analysis was performed with ClueGO. Results: Chromosomal gains and losses were confirmed to be a preponderant feature of COMs, with a particular sigmoidal trend for aberration in CFA 30 and 10. We confirmed some CNAs recently reported and described new unreported chromosomal rearrangements. Many aberrant regions overlapped with HMM-associated CNAs. Statistical analyses revealed imbalances of MAPK- and PI3K-related genes, and the perturbation of cancer-related biological processes, in particular neoangiogenetic pathways. Conclusions: Our data provide a new insight into COM biology with detection of genes and pathways related to angiogenesis, cell proliferation and immune response. Neovascularisation, described in other human melanomas, is essential for primary tumour and metastasis survival, revealing this to be a promising target for future studies aimed to deepen understanding of the pathogenesis of COM

Clinical and histopathological characteristics of two canine gastric vascular hamartomas

Canine gastrointestinal hamartomas are rare presentations that have only been detected once with a gastric location, in a case of smooth muscle hamartoma [1]. Like other diseases causing an encumbrance on the gastric lumen, they can clinically manifest with gastrointestinal signs [2]. Two elderly mixed breed dogs presented with chronic vomit and lack of appetite, with ultrasound and tomographic evidence of an intraluminal gastric mass of unknown origin. In both cases, the masses were surgically resected and sent to the histopathology service of the Department of Comparative Biomedicine and Food Science. The two masses appeared macroscopically as either an exophytic mucosal and submucosal polypoid proliferation affecting the fundic region (dog 1), or as a mural submucosal bulging mass from the pyloric region (dog 2). After fixation and processing, the histological slides were evaluated by hematoxylin-eosin (HE) staining. Sub-grossly, the polypoid mass (dog 1) was poorly demarcated, in contrast to the mass of dog 2. Microscopically, both showed an irregular proliferation of vascular structures considered veins and arteries, severely expanding the submucosa in both cases and displacing the tunica muscularis in one case (dog 2). The vessels were lined by factor VIII+ non-atypical endothelial cells, often supported by irregularly thickened and shaped, muscular, α-SMA+ walls. The vessels were often engorged by red blood cells and multiple intravascular thrombi were noted. The polypoid mass also showed a mild lymphocytic infiltration of the adjacent mucosa and submucosa, while the second mass had a necrotic center with dystrophic mineralization and neutrophilic inflammation. They have been diagnosed as a gastric hamartomatous polyp of vascular type (dog 1) and as a gastric vascular hamartoma (dog 2). Both dogs recovered well and were healthy one year after the surgery. Dog 1 is still alive, while dog 2 was euthanized for unrelated causes. In both cases, complete surgical excision was conclusive for diagnosis and treatment. This approach is indeed preferred to endoscopic biopsy, allowing the evaluation of the deeper layers. In conclusion, the two masses had a different macroscopic presentation that initially led to consider them two different entities. However, on microscopic examination, both showed a prominent proliferation of well-developed, but disorganized, non-infiltrating vascular structures characterized by strong immunolabeling for factor VIII and α-SMA protein. These findings led to a common diagnosis of gastric vascular hamartoma. Due to their good prognosis, hamartomas should be considered among the differential diagnoses and differentiated from neoplasms with which they can share the macroscopic presentation. [1] Taulescu et al. Gastric smooth muscle hamartomas mimicking polyps in a dog: A case description and a review of the literature. Case Rep. Vet. Med., 2013: 1–5, 2013. [2] Diana A, Penninck DG, Keating JH. Ultrasonographic appearance of canine gastric polyps. Vet. Radiol. Ultrasound, 50: 201-204, 2009

The use of Corticotropin-releasing factor (CRF) as a marker of agonic stress in abused dogs: Preliminary results

In Veterinary Forensic Medicine, determination of the degree of animal suffering is an essential element for the prosecution of perpetrators of animal abuse. The purpose of this study is to find a suitable immunohistochemical marker for the assessment of suffering to be routinely used in Veterinary Forensic Pathology, by analyzing the expression of Corticotropin-releasing factor (CRF) in formalin-fixed brains of dogs as a measurement of the agonic stress. CRF, a key peptide element in exogenous and endogenous stressors adaptation, can regulate endocrine-behavioral responses to stress stimulating pituitary ACTH release and consequent adrenal secretion of glucocorticoids. Since CRF acts in days or weeks, this study investigates its role as a potential distinctive marker between sudden death and death associated with a longer agonic period. The study used immunohistochemistry (IHC) to evaluate the CRF expression in the brain of dogs that suffered sudden death, as compared to dogs that died after long-term agonic stress. IHC labelling analysis was performed with machine-learning-based software and the results were statistically evaluated. Our results demonstrate for the first time that CRF is a promising marker of stress in abused patients also in Veterinary Medicine