Digital Measurement of the Diameter of the Collagen Fibers by Transmission Electron Microscopy for Diagnosis of Ehlers-Danlos Syndrome-Like in Small Animals

Background: Ehlers-Danlos-Like Syndrome (EDS) is a rare disease in small animals, whose diagnosis is based on the clinical findings and histopathological examination. The definitive diagnosis may require transmission electron microscopy. Despite this, the ultrastructural changes are poorly described in the literature. The aim of the present study is to describe the ultrastructural findings of collagen fibers and fibroblasts present in the dermis of two animals with EDS, and to evaluate the digital measurement of the diameter of the collagen fibers by transmission electron microscopic images.Cases: Two animals were evaluated with EDS by transmission electron microscopy. The first animal was an 1-year-old mixed breed female cat, due to spontaneous skin laceration, increased skin elasticity and an extensibility index corresponding to 25%. The second animal evaluated was a 5-month-old Golden Retriever female dog due to articular hypermobility, increased skin elasticity and an extensibility index corresponding to 16.6%. After a skin biopsy of the interscapular and lumbar regions, the samples were fixed in formalin 10% and glutaraldehyde for, respectively, histopathological examination by HE staining, and transmission electron microscopy. The histopathology of the affected cat revealed collagen fibers shortened and sometimes fragmented. The histopathology of the affected dog revealed disarranged and more eosinophilic staining collagen fibers. The collagen fibers were also of unequal sizes, shortened and slightly undulate. At the transmission electron microscopy of the affected cat was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, the fibroblasts showed elongated nuclei with heterochromatic regions, which was surrounded externally by scant cytoplasm. The cytoplasm showed elongated and discrete organelles. At the transmission electron microscopy of the affected dog was evidenced a greater spacing of the collagen fibers of variable diameters. Further this, fibroblasts exhibited intense cytoplasmic vacuolization with similar appearance to that found in the dying process by autophagy. In addition, the images obtained by transmission electron microscopy were submitted to digital analysis to measure the diameter of the collagen fibers using the software Image J. For this purpose, it was obtained the average of the diameter of 10 collagen fibers in cross-section into four quadrants of 1μm each. The digital analysis of collagen fibers revealed significant alterations in the ultrastructure of collagen. In addition, it was verified cellular changes, such as the large amount of intracytoplasmic vesicles and the small amount of collagen fibers dispersed around the cells.Discussion: Microscopic abnormalities visualized by HE staining in this present study were compatible with the literature Transmission electron microscopy is fundamental to confirm the suspicion, since it revealed dermal alterations in the ultrastructure of collagen and fibroblasts. These findings indicate possible failure mechanisms of secretion and release of cellular products, as well as collagen. The digital measurement of the diameter of the collagen fibers contributed to the confirmation of the disease, since it was made randomly and reduced the subjectivity inherent of the evaluator. However, there are no studies using this method to allocate a range of significance for the diameter of collagen fibers that may be considered suggestive for the syndrome in small animals

Fauna invertebrada e atributos físicos e químicos do solo em sistemas de integração lavoura‑pecuária

The objective of this work was to evaluate the effect of integrated crop-livestock (ICL) systems on the main groups of soil invertebrate macrofauna and the relationship of these organisms with soil physical and chemical attributes. The conventional system, integrated crop-livestock system, no-tillage system, continuously cultivated pasture, and natural fragments of Cerrado and of semi-deciduous forest were compared. The experiment was carried out in Dourados, MS, Brazil, on a Typic Hapludox soil, divided into strips. The highest density, richness, and diversity of families were found in the ICL system, under no-tillage, and in the continuously cultivated pasture. The conventional system caused greater reduction of the soil invertebrate community. The ICL system favors the maintenance of the invertebrate fauna diversity, the formation of stable aggregates, and soil fertility. The Sthaphylinidae family is a potential soil quality bioindicator, especially in the semi-deciduous forest and in the ICL system.O objetivo deste trabalho foi avaliar o efeito do sistema de integração lavoura-pecuária (ILP) nos principais grupos da macrofauna invertebrada do solo e a relação destes organismos com os atributos físicos e químicos do solo. Foram comparados: sistema de manejo convencional, sistema integração lavoura-pecuária, sistema plantio direto, pastagem cultivada continuamente e fragmentos naturais de Cerrado e de floresta semidecídua. O experimento foi conduzido em Dourados, MS, em um Latossolo Vermelho distroférrico típico, disposto em faixas. A maior densidade, riqueza e diversidade das famílias foi observada no sistema de ILP, em plantio direto, e no sistema em pastagem cultivada continuamente. O sistema convencional promoveu maior redução na comunidade dos organismos invertebrados do solo. O sistema de ILP favorece a manutenção da diversidade da fauna invertebrada, a formação dos agregados estáveis e a fertilidade do solo. A família Sthaphylinidae é um potencial bioindicador de qualidade do solo, principalmente na floresta semidecídua e no sistema de ILP

Histomorphometric analysis of the Achilles tendon of Wistar rats treated with laser therapy and eccentric exercise

Abstract: Low-level laser therapy is recommended for the treatment of tendinopathies despite the contradictory results related to the ideal dose of energy, wavelength and time of application. This study aimed to assess the effects of laser therapy and eccentric exercise on tendinopathy of the Achilles tendon of Wistar rats. Forty-eight adult male rats were randomly distributed into four groups (L= laser; E= eccentric exercise; LE = laser and eccentric exercise; and R= rest). Laser therapy (904nm/3J/cm2) and/or eccentric exercise (downhill walking; 15o incline treadmill; 12m/min; 50min/day) was started 24h after induction of unilateral tendinopathy and remained for 20 days. At 3, 7, 14 and 21 days after lesion induction, three rats from each group were euthanized and the tendons were collected for histological and morphometric analyses. There was no difference among groups or among times for the characteristics hemorrhage (p=0.4154), fibrinous adhesion formation (p=0.0712), and organization of collagen fibers (p=0.2583) and of the connective tissue (p=0.1046). For these groups, regardless of the time, eccentric exercise led to epitenon thickening (p=0.0204), which was lower in the group treated with laser therapy. Histological analysis revealed differences (p=0.0032) in the number of inflammatory cells over time. They were more numerous in the group that only exercised. This result was confirmed by morphometric analysis, which showed a significant interaction (groups x time) for this characteristic. Eccentric exercise increased (p=0.0014) the inflammatory infiltrate over time (3 and 21 days). However, association with laser therapy reduced inflammatory reaction. On the other hand, the combination of the treatments increased angiogenesis in morphometric (p=0.0000) and histological (p=0.0006) analyses compared with the other groups, while the isolated application of low-level laser reduced this characteristic over time. Animals maintained at rest presented the lowest amount (p=0.0000) of fibroblasts, according to the morphometric analysis. However, histological evaluation showed a significant group x time interaction (p=0.0024). Greater amounts of fibroblasts were observed in groups E, L and LE on the 7th, 14th and 21st days, respectively. The animals that received laser therapy and were exercised showed a greater (p=0.0000) amount of collagen fibers over time. Laser therapy at a dose of 3J and at a wavelength of 904nm, starting 24h after surgical induction of tendinopathy in Wistar rats, is suitable for angiogenesis and prevention of tendon thickening, which can be associated with the intensity of inflammatory process. When associated with eccentric exercise, the therapy has the advantage of increasing the amount of collagen fibers, reducing fibrinous adhesions and inflammatory infiltrate, despite prolonging angiogenesis. Therefore, eccentric exercise performed concomitantly with laser therapy improves the histological properties of the injured tendon

Immunohistochemical Expression of Collagens in the Skin of Horses Treated with Leukocyte-Poor Platelet-Rich Plasma

This study evaluated the immunohistochemical expression of type I (COL I) and III (COL III) collagens during the healing process of skin treated with leukocyte-poor platelet-rich plasma (LP-PRP). Seven healthy gelding crossbred horses aged 16 to 17 years were used. Two rectangle-shaped wounds were created surgically in the right and left gluteal regions. Twelve hours after wound induction, 0.5 mL of the LP-PRP was administered in each edge of the wounds of one of the gluteal regions. The contralateral region was used as control (CG). Three samples were obtained: after wound induction (T0), 14 days (T1) of healing process, and after complete closure of the skin (T2). The normal skin (T0) showed strong staining for type III and I collagen in papillary and reticular dermis, respectively. In the scar of the treated group, COL III showed important (p<0.05) increase in immunoreaction in T2 compared with T1. The administration of a single dose of LP-PRP 12 h after induction of wound in horses does not influence formation of collagens I and III. However, the intense labeling for COL III suggests that the tissue was still weak during the macroscopic closure of the wound, demonstrating that healing was not completely finished

Fauna invertebrada e atributos físicos e químicos do solo em sistemas de integração lavoura-pecuária.

O objetivo deste trabalho foi avaliar o efeito do sistema de integração lavoura-pecuária (ILP) nos principais grupos da macrofauna invertebrada do solo e a relação destes organismos com os atributos físicos e químicos do solo. Foram comparados: sistema de manejo convencional, sistema integração lavoura-pecuária, sistema plantio direto, pastagem cultivada continuamente e fragmentos naturais de Cerrado e de floresta semidecídua. O experimento foi conduzido em Dourados, MS, em um Latossolo Vermelho distroférrico típico, disposto em faixas. A maior densidade, riqueza e diversidade das famílias foi observada no sistema de ILP, em plantio direto, e no sistema em pastagem cultivada continuamente. O sistema convencional promoveu maior redução na comunidade dos organismos invertebrados do solo. O sistema de ILP favorece a manutenção da diversidade da fauna invertebrada, a formação dos agregados estáveis e a fertilidade do solo. A família Sthaphylinidae é um potencial bioindicador de qualidade do solo, principalmente na floresta semidecídua e no sistema de ILP.201