P–452 The challenge of ovarian tissue culture: 2D versus 3D

Study question Does an alginate matrix scaffold improve ovarian tissue culture? Summary answer Ovarian tissue culture within an alginate scaffold has no advantage over conventional culture, being more time consuming and less reproductible What is known already Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. Several studies have been carried out aiming to increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells. For in vitro follicle culture, recent research has shifted from two dimensional (2D) toward the use of three-dimensional (3D) structures. The use of a matrix maintains the architecture and mimics in vivo conditions, with a variable access to oxygen and nutrients. This bridges the gap between conventional cell culture and animal models. Study design, size, duration Ovarian tissue fragments were divided into 2 groups: conventional culture (2D culture) and culture using an alginate matrix scaffold (3D culture). Tissue was evaluated at four time-points: immediately after thawing and after 24, 48 and 72 hours of culture. Participants/materials, setting, methods Rat ovarian tissue was cryopreserved and thawed with validated protocols. Follicular analysis was conducted after haematoxylin and eosin staining, regarding density, classification and degeneration. Tissue viability was assessed using lactate dehydrogenase (LDH) levels in supernatants and histological score. Three parameters were considered, namely, interstitial oedema, follicular cell degeneration and percentage of tissue in necrosis. Apoptosis was assessed by caspase 3 immunostaining. Proliferating cells were identified using Ki67 immunohistochemical labelling. Main results and the role of chance Follicular density, cell proliferation and apoptosis both in follicles and stroma was similar in both culture conditions. Stromal cells proliferation was stable in conventional culture but decreased in 3D culture (p = 0.001), which can be explained by the rigidity of alginate matrix. At 24 hours of culture, cytotoxicity was lower in the 3D model (p = 0.006), due to low levels of LDH in the supernatant, that may be related to retention within the matrix. As culture time increased cytotoxicity seemed to be similar. Degradation of the tissue was suggested by the histological score analysis of tissue during 72 hours of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). In the interior of the alginate scaffold, the bioavailability of oxygen and nutrients may be limited, affecting cell survival over time and conditioning higher level of necrosis and release of intracellular content. Limitations, reasons for caution There are two major limitations that should be addressed in future research, namely the study of the tissue-matrix interactions and culture medium supplementation to decrease follicular atresia. Wider implications of the findings: There is no advantage in the use of an alginate matrix scaffold for ovarian tissue culture, as it is more time consuming, difficult to perform and less reproductible. Trial registration number Not applicable

Acute and severe haemorrhage following pentosan polysulfate injection in a Cornish Rex

Case summary A 14-year-old male castrated Cornish Rex cat was referred for lethargy progressing rapidly to collapse in the hours following a subcutaneous injection of a product containing 100 mg/ml pentosan polysulfate sodium and 168 mg/ml glucosamine. Physical examination revealed the cat to be in hypotensive shock with swelling and interstitial oedema around the cranial thorax and caudal cervical regions without cutaneous haemorrhage. Initial diagnostics revealed a severe anaemia (packed cell volume 11%) which later deteriorated further, necessitating a blood transfusion and aggressive fluid therapy. Post-transfusion, the patient remained dyspnoeic and subsequent diagnostics found evidence of pre-existing cardiomyopathy and congestive heart failure. The cat was euthanased 24 h following presentation due to increasing dyspnoea. Post-mortem findings were of severe subcutaneous and intermuscular haemorrhage over the neck and thorax, among other changes. There were no detectable levels of coumarin anticoagulants in the liver. Relevance and novel information This is the first reported case of acute subcutaneous and intermuscular haemorrhage of this severity suspected to be related to the off-label use of an injectable product containing pentosan polysulfate in a cat. Given the popularity of its use for feline arthritis, there is a need for large-scale clinical trials to evaluate the safety and efficacy of products containing pentosan polysulfate for cats, and any side effects to be reported.

Cardiomyopathies and Adrenal Diseases

Cardiomyopathies are myocardial disorders in which heart muscle is structurally and/or functionally abnormal. Previously, structural cardiomyocyte disorders due to adrenal diseases, such as hyperaldosteronism, hypercortisolism, and hypercatecholaminism, were misunderstood, and endomyocardial biopsy (EMB) was not performed because was considered dangerous and too invasive. Recent data confirm that, if performed in experienced centers, EMB is a safe technique and gives precious information about physiopathological processes implied in clinical abnormalities in patients with different systemic disturbances. In this review, we illustrate the most important features in patients affected by primary aldosteronism (PA), Cushing’s syndrome (CS), and pheochromocytoma (PHEO). Then, we critically describe microscopic and ultrastructural aspects that have emerged from the newest EMB studies. In PA, the autonomous hypersecretion of aldosterone induces the alteration of ion and water homeostasis, intracellular vacuolization, and swelling; interstitial oedema could be a peculiar feature of myocardial toxicity. In CS, cardiomyocyte hypertrophy and myofibrillolysis could be related to higher expression of atrogin-1. Finally, in PHEO, the hypercontraction of myofilaments with the formation of contraction bands and occasional cellular necrosis has been observed. We expect to clear the role of EMB in patients with cardiomyopathies and adrenal disease, and we believe EMB is a valid tool to implement new management and therapies.

Experimental isolation and preservation of solid organs before transplantation: effects of pretreatment using four different molecules

Objectives In transplantation surgery, the ischaemic organ and reperfusion impairment after cold storage remains a considerable risk factor for impaired function and potential failure of the grafted organ. Substantial logistical efforts have been undertaken to reduce the cold ischaemic time because the demand for available transplant organs and the periods of cold ischaemia are increasing. Methods Four molecules were investigated (erythropoietin, sildenafil, lazaroid [U74389G], octreotide) in individual intravenous infusions 1 hour before the organ was harvested. This study was performed in 30 healthy landrace/large-white pigs (male; >10 weeks old; average weight, 22 ± 2 kg) in groups of six. The organs were studied at harvest, and at 8 and 24 hours post-harvest. Results The lazaroid molecule increased malondialdehyde (MDA) levels in the liver and pancreas at 8 hours. Hepatic lazaroid molecules improved liver histology at 8 and 24 hours. For kidneys, erythropoietin had a positive effect at 24 hours post-harvest. For the pancreas, octreotide showed better performance. In the lungs, there was less interstitial oedema with erythropoietin and lazaroid compared with the control group at 8 hours post-harvest. Conclusion All molecules had a positive effect and decreased ischaemia/reperfusion graft injury. Thus, pretreatment before organ harvest has a beneficial role.

Peripheral oedema

Peripheral oedema is a palpable swelling caused by increased interstitial fluid in soft tissues, and can be due to local or systemic disease. Fluid distribution between capillaries and the interstitium is governed by Starling forces. The lymphatic system returns excess fluid and protein from the extracellular, interstitial space to the bloodstream. Thus, interstitial oedema may arise from factors that increase capillary pressure or permeability, factors that reduce plasma colloid osmotic pressure, factors that impede lymphatic drainage, or a combination of these causes. This topic addresses the diagnosis of peripheral oedema.

Subclinical pulmonary edema in endurance athletes

Strenuous exercise may cause progressive and proportional haemodynamic overload damage to the alveolar membrane, even in athletes. Despite the high incidence of arterial desaturation reported in endurance athletes has been attributed, into other factors, also to the damage of the alveolar-capillary membrane this evidence is equivocal. Some studies demonstrated flood of the interstitial space and consequent increase in pulmonary water content, but most of them were able to show this through indirect signs of interstitial oedema. The present review illustrates the literature’s data in favour or against pulmonary interstitial edema due to intense exercise in athletes.

Microscopic changes in the kidneys of cows infected with Leptospira sp.

The article presents data on histopathological studies of the kidneys of cows, which either recovered or died from leptospirosis. Fragments of seven kidneys from slaughtered cows, positive for Leptospira antibodies in the microscopic agglutination test (MAT) (titres of 50 and higher) were used in the study. The MAT was conducted with eight serological groups of Leptospira: Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Pomona, Sejroe, Tarassovi, and Australis. Microscopic changes in all morphological structures of the kidneys were presented. Micromorphological criteria, which can be used for post-mortem diagnosis of leptospirosis were established. They included: serous glomerulonephritis with granular dystrophy of podocytes, necrosis and collapse of the inner layer of Bowman's capsule, partial destruction of capsule and vascular glomeruli, granular and vacuolar degeneration and destruction of tubular epithelial cells, foci of interstitial oedema, and infiltrations predominantly with monocytes and isolated neutrophils. Microscopic changes in the kidneys suggest that the cows died from leptospirosis as a result of toxic shock syndrome.

Histopathological and haematological response of male rainbow trout (Oncorhynchus mykiss) subjected to butachlor

This study was aimed at assessing the histopathological and haematological effects of a widely used herbicide on rice paddy fields, i.e. butachlor, on rainbow trout. Fish were exposed to butachlor at a concentration of 0.39 mg/l, for a period of 10 days. Haematologically, fish showed a significant decrease in erythrocyte count, haemoglobin, white blood cells and lymphocytes and a significant increase in neutrophils compared to controls (P < 0.05). Histopathological observations of prepared sections of the treatment group also revealed pathological lesions of varying severity in studied organs, including liver (hyperaemia and haemorrhage, bile duct hyperplasia, dilated sinuses, interstitial oedema, monocellular necrosis, nuclear degeneration and hypertrophy in hepatocytes), gills (hyperplasia and hyperplasia of lamellar epithelium, fusion of lamellae, rod-like structures of secondary gill lamellae, cystic-like lesions) and kidneys (vacuolar degeneration of tubular epithelium, desquamation of epithelium and necrosis of tubular epithelium). It is concluded that butachlor caused changes in certain haematological parameters and histopathologically, exerted destructive effects on the gills, liver and kidneys of rainbow trout.