Cryodehydration applied to bovine fetal hearts: A model for studying cardiac organogenesis.

The study aimed to standardize the cryodehydration technique for bovine fetal hearts, focusing on optimizing protocols for each developmental stage to preserve morphological characteristics. We analyzed 29 bovine fetal hearts categorized into early, middle, and late developmental stages. These hearts underwent cryodehydration until a 60%-70% reduction in original fluid volume was achieved. Biometric data were recorded and statistically analyzed using Pearson correlation tests for age versus weight and age versus number of cryodehydration sessions. Morphometric comparisons before and after cryodehydration were performed using paired t-tests. In Group I, hearts exhibited well-defined structures, including the atrium cordis, ventriculus cordis, auricula atrii, aorta, truncus pulmonalis, and ramus coronaries arteria, which were preserved in Groups II and III. Additionally, in Group I the heart had a conical or flat apex cordis, whereas those in Groups II and III had a more pronounced apex. The average number of cryodehydration sessions required was 9.38 (±1.2) days for Group I, 12.37 (±1.4) days for Group II, and 15 days for Group III. A positive correlation was found between age and sample weight, indicating that more developed hearts were heavier. Similarly, there was a positive correlation between gestational age and the number of cryodehydration sessions, suggesting that more advanced stages required more cryodehydration sessions. Paired t-tests demonstrated high statistical significance in the morphometric parameters before and after cryodehydration, indicating a loss of mass during dehydration. However, there was no alteration in the macroscopic structure of the hearts, which remained morphologically preserved. In conclusion, cryodehydration shows promise for preserving and analyzing the external morphological characteristics of bovine fetal cardiac development. It also provides lightweight, odorless, and easy-to-handle specimens ideal for detailed morphological studies and offers a unique perspective for investigating cardiac morphology in biological research contexts.

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation.

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin-eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.

Immunophenotyping of progenitor cells from articular cartilage of New Zealand Rabbits (Oryctolagus cuniculus).

Mesenchymal stem cell (MSC) have immunomodulatory and anti-inflammatory effects, allowing its application in the therapy of different diseases, including articular cartilage injuries, which induce the establishment of a pro-regenerative microenvironment in the injured tissue. Therefore, our objective was to isolate, characterize and differentiate cartilage cells from different joints of New Zealand rabbit (Oryctolagus cuniculus), in order to verify their potential as MSC for future clinical use. For this, cartilage fragments were isolated from the humerus-radio-ulnar joints, humeral scapula, femoro-tibio-patellar, and lame femoris from rabbits. The results showed that the cells were rounded in the center of the plate and fibroblastoids in the periphery. After thawing, the cells did not change their growth time in culture, nor their morphology. The cells showed labeling for mesenchymal stem cell, cytoskeleton, pluripotency and cell proliferation, but not for hematopoiesis markers (CD105+ and CD34-). We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. After application of these cells in nude mice, no tumor growth was observed in spleen, kidney, liver, lung and heart. Therefore, we conclude that cells isolated from the articular cartilage of rabbits present characteristics of MSC with potential for future clinical applications.

Central nervous system development in rabbits (Oryctolagus cuniculus L. 1758).

The present study describes the embryonic and fetal development of the central nervous system in rabbits from the seventh day after conception until the end of the full-term fetal period. A total of 19 embryonic and fetal samples were carefully dissected and microscopically analyzed. Neural tube closure was observed between 7.5 and 8 days of gestation. Primordial encephalic vesicle differentiation and spinal canal delimitation were observed on the 12th day of gestation. Histologically, on the 15th day of gestation, the brain, cerebellum, and brain stem were delimited. On the 18th day of gestation, the cervical and lumbar intumescences of the spinal cord were visible. On the 28th day of gestation, four-cell layers could be distinguished in the cerebral cortex, while the cerebellar cortex was still differentiating. Overall, the morphological aspects of the embryonic and fetal developmental phases in rabbits were highly similar to those in humans. Thus, the present study provides relevant information highlighting rabbits as an excellent candidate animal model for preclinical research on human neurological diseases given the high adaptability of rabbits to bioterium conditions and the similarity of morphological events between rabbits and humans.

Evaluation of immunohistopathological profile of tubular and solid canine mammary carcinomas.

Breast cancer is the most common cancer in women, but the incidence of mammary carcinoma in female dogs is even higher than in humans. These two tumors have similarities that can be seen by its biological behavior, molecular genetic alterations, and histology. This suggest that female dogs can be an excellent model for preclinical oncological studies. And the mammary carcinoma most frequently found in this species is the tubular and solid carcinomas. The extracellular matrix (ECM) has an important role in the progression of these tumors. Because of that we proposed to evaluate the ECM components of these carcinomas through histology with specific stains such as Masson's Trichrome, Picrosirius Red and the technique of scanning electron microscopy. With that, we found the presence of collagen fibers in the tubular carcinoma and around its parenchyma. On the other hand, the solid carcinoma presented collagen fibers throughout the parenchyma and around each tumor cell. With the transmission electron microscopy, we observed the presence of mitochondrias and rough endoplasmic reticulum in both tumors. And finally, we evaluated the expression of proteins through the immunohistochemistry, in which we found a high expression of VEGF, PCNA, CK-18 and vimentin in solid carcinoma, and a positive mark in the tubular and solid carcinoma for collagen I, III and fibronectin. Thus, we demonstrated some differences in the ECM of these mammary carcinomas, allowing a better understanding of its histological characteristics, and these data may contribute to future studies about therapies focused on tumors ECM.

Canine amniotic membrane mesenchymal stromal/stem cells: Isolation, characterization and differentiation.

The amniotic membrane can be considered as one of the sources of isolation of these cells, since it is found in the fetal maternal interface and has low immunogenicity. Mesenchymal stromal/stem cells (MSCs) have not been identified in canine amniotic membrane (AMC). Therefore, our objective was to isolate, culture, characterize and differentiate cells derived from canine amniotic membrane (AMC) and to verify its immunological and tumorigenic potential. For this, 12 dogs fetuses of each gestational age 32, 43 and 55 days were used, and the isolation and culture of the AMC were performed. We observed that the cells presented fibroblastoid morphology and high confluence even after freezing. We also observed that, when induced, they were able to differentiate into osteogenic, adipogenic, and chondrogenic cells, as well as being CD34- and CD105+. Regarding the immunological markers, we found that IL-1, IL-2, IL-6, IL-10 and MHC II were not expressed, whereas MHC I was expressed. After application of AMC cells in nude mice we can verify that there was no tumor formation. Based on this, we conclude that canine amniotic membrane is a good and accessible source for obtaining MSCs of low immunogenic and tumorigenic potential for veterinary therapeutic applications.