APLICACIÓN DE COLORACIONES ESPECIALES EN EL DIAGNÓSTICO HISTOPATOLÓGICO: TINCIÓN TRICRÓMICA DE MASSON

Masson's trichrome (MT) is a three-color staining protocol used in histology. MT allows to show and quantify changes such as tissue repair (healing) and collagen deposition. Also, it can be used to quantify blood vessels, in epithelial dysplasia and squamous cell carcinoma. The objective of this work is to describe the MT staining technique and to exemplify some applications of this technique in routine veterinary histopathological diagnosis. Archived histologic sections were selected from the records of the histopathology laboratory. Tissues were selected in base on theirs structures and lesions that could be evaluated with MT: a rabbit lung with a chronic suppurative bronchopneumonia; a bovine liver with lesions of Echium plantagineum poisoning; and a bovine eyelid with a squamous cell carcinoma. The TM was able to show fibroplasia in the pulmonary interstitium and confirm the presence of a chronic respiratory process, and clearly revealed an abundant fibrovascular tumor stroma, with profuse connective tissue and neovascularization between the tumor cells in deep dermis. In the liver, extensive and marked fibroplasia was confirmed. MT represents a complementary coloration to routine hematoxylin and eosin stain (H&E) and provides accurate information from several pathological processes, mainly those related to fibrovascular proliferation and scarring.

Characterization of microRNA Profiles in Pasteurella multocida-Infected Rabbits and Identification of miR-29-5p as a Regulator of Antibacterial Immune Response

Pasteurella multocida is the pathogenic agent for a variety of severe diseases in livestock, including rabbits. MicroRNAs (miRNAs) participate in the immune response to the pathogen. Distinct miRNA expression patterns were explored in rabbit lung by small-RNA deep sequencing to assess dysregulated miRNAs during P. multocida infection. Totally, 571 miRNAs were screened, of which, 62 were novel, and 32 exhibited differential expression (DE). Of the 32 known DE-miRNAs, 13 and 15 occurred at 1 day and 3 days post-infection (dpi); and ocu-miR-107-3p and ocu-miR-29b-5p were shared between the two time points. Moreover, 7,345 non-redundant target genes were predicted for the 32 DE-miRNAs. Putative target genes were enriched in diverse GO and KEGG pathways and might be crucial for disease resistance. Interestingly, upregulation of ocu-miR-29-5p suppresses P. multocida propagation and downregulates expression of epithelial membrane protein-2 (EMP2) and T-box 4 (TBX4) genes by binding to their 3′ untranslated region in RK13 cells. Thus, ocu-miR-29-5p may indirectly inhibit P. multocida invasion by modulating genes related to the host immune response, such as EMP2 and TBX4.

Lung ultrasound features and relationships with respiratory mechanics of evolving BPD in preterm rabbits and human neonates

Evolving broncho-pulmonary dysplasia (BPD) is a regionally heterogeneous disorder characterized by impaired alveolarization leading to lung aeration inhomogeneities. Hyperoxia-exposed preterm rabbits have been proposed to mimic evolving BPD and we aim to verify if this model has the same lung ultrasound and mechanical features of evolving BPD in human neonates. Twenty-five preterm rabbits and twenty-five neonates with evolving BPD were enrolled and subjected to semi-quantitative lung ultrasound and lung mechanics measurement. A modified rabbit lung ultrasound score (rLUS), the previously validated neonatal lung ultrasound score (LUS) and classical mechanics measurements were obtained. Lung ultrasound images were also recorded and evaluated by two independent observers with different expertise blinded to each other's evaluation. Lung ultrasound findings were equally heterogeneous both in rabbits as in human neonates: images were very similar and encompassed all the classical lung ultrasound semiology. The inter-rater absolute agreement for the evaluation of lung ultrasound images in rabbits was very high (ICC: 0.989 (95%CI: 0.975-0.995); p<0.0001) and there was no difference between the two observers. Lung mechanics parameters were similarly altered both in rabbits and human neonates. There were significant correlations between airway resistances and lung ultrasound scores both in rabbits (r=0.519; p=0.008) and in neonates (r=0.409; p=0.042). No significant correlation between rLUS, LUS and any other mechanics parameter. Lung ultrasound was easy to be performed and accurate even in these small animals and with a short training. In conclusion, the preterm rabbit model fairly reproduces the lung ultrasound and mechanical characteristics of preterm neonates with evolving BPD.

In vivo tumour imaging employing regional delivery of novel gallium radiolabelled polymer composites

Background Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. Results Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 μm diameter MS than with 30 μm MS, and the smaller microspheres provided more distinct and localised tumour imaging. Conclusion This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.

Combination of µCT and light microscopy for generation-specific stereological analysis of pulmonary arterial branches: a proof-of-concept study

Various lung diseases, including pulmonary hypertension, chronic obstructive pulmonary disease or bronchopulmonary dysplasia, are associated with structural and architectural alterations of the pulmonary vasculature. The light microscopic (LM) analysis of the blood vessels is limited by the fact that it is impossible to identify which generation of the arterial tree an arterial profile within a LM microscopic section belongs to. Therefore, we established a workflow that allows for the generation-specific quantitative (stereological) analysis of pulmonary blood vessels. A whole left rabbit lung was fixed by vascular perfusion, embedded in glycol methacrylate and imaged by micro-computed tomography (µCT). The lung was then exhaustively sectioned and 20 consecutive sections were collected every 100 µm to obtain a systematic uniform random sample of the whole lung. The digital processing involved segmentation of the arterial tree, generation analysis, registration of LM sections with the µCT data as well as registration of the segmentation and the LM images. The present study demonstrates that it is feasible to identify arterial profiles according to their generation based on a generation-specific color code. Stereological analysis for the first three arterial generations of the monopodial branching of the vasculature included volume fraction, total volume, lumen-to-wall ratio and wall thickness for each arterial generation. In conclusion, the correlative image analysis of µCT and LM-based datasets is an innovative method to assess the pulmonary vasculature quantitatively.

Marathoners' Breathing Pattern Protects Against Lung Injury by Mechanical Ventilation: A Pilot Study

Background Breathing during a marathon is often done empirically in a so-called “2:2 breathing rhythm”. This breathing rhythm is based on cycles composed of four phases: the 1st inspiratory period, 2nd inspiratory period, 1st expiratory period, and, finally, the 2nd expiratory period. We developed a prototype ventilator that can perform intermittent positive pressure ventilation, mimicking the breathing cycle of the 2:2 breathing rhythm. This mode of ventilation was named the marathoners’ breathing rhythm ventilation (MBV) and we hypothesized that MBV may have a lung protective effect.Methods We examined the effects of the MBV on the pulmonary pre-edema model in isolated perfused rabbit lungs. The pulmonary pre-edema state was induced using bloodless perfusate with low colloid osmotic pressure. The fourteen isolated rabbit lung preparations were randomly divided into the inversed ratio ventilation (IRV) group and the MBV group, (both had an inspiratory:expiratory ratio of 1:1). In the IRV group, seven rabbit lungs were ventilated using a Harvard Ventilator 683 with a tidal volume (TV) of 8 mL/kg, a respiratory rate (RR) of 30 cycles/min, and a positive-end expiratory pressure (PEEP) of 2 cmH2O for 60 min. In the MBV group, seven rabbit lungs were ventilated using the prototype ventilator with a TV of 6 mL/kg, an RR of 30 cycles/min, and a PEEP of 4 cmH2O (first step) and 2 cmH2O (second step) for 60 min. The time allocation of the MBV for one cycle was as follows: 1st inspiratory period, 2nd inspiratory period, 1st expiratory period, and 2nd expiratory period (all 0.3 s long), with intermittent resting periods (all 0.2 s).Results Peak airway pressure and lung wet-to-dry ratio after 60 min ventilation were lower in the MBV group than in the IRV group.Conclusion MBV was considered to have a lung protective effect compared to the IRV method.

The study of angiotensin converting enzyme isolation from crossbred rabbit lung and enzyme storage capacity in frozen conditions

The study was carried out to obtain angiotensin converting enzyme (ACE) with high specific activity and to evaluate the ability to maintain enzyme activity in extract products as well as in rabbit lungs using frozen condition. In the scope of the content, the study conducted an evaluation to select the appropriate extraction solvent of four solvents including acetone, ethanol, Tris-HCl and distilled water. Initially, the research results have helped determine distilled water as the suitable extraction solvent and the difference is not statistically significant compared to Tris-HCl solvent. Angiotensin converting enzyme extract that was obtained by distilled water solvent has a specific activity of 10.35 U/g protein. In addition, the study investigated the ability to maintain angiotensin converting enzyme activity in rabbit lung and crude enzyme product during frozen storage (-18±2°C). The results of the study showed that angiotensin converting enzyme activity could be maintained for 3 months in rabbit lungs and 4 months in the crude product. Besides, the study also used ammonium sulfate with different concentrations to conduct angiotensin converting enzyme collection from extract product. The results of this content help determine the use of saturated ammonium sulfate with concentrations of 50% to 60% for the highest efficiency. The precipitation process helped obtain ACE products with a purity of 4.22 times, the specific activity of 42.64 U/g protein and the recovery rate of ACE up to 29.37%.