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Einstein (Säo Paulo) ; 20: eAO6450, 2022. tab, graf
Article in English | LILACS | ID: biblio-1364794


ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Urinary Bladder Neoplasms/genetics , Carcinoma, Transitional Cell/genetics , Carcinoma, Transitional Cell/metabolism , Carcinoma, Transitional Cell/pathology , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Brazil , RNA , Prevalence , Eosine Yellowish-(YS) , Hematoxylin , Muscles/metabolism , Muscles/pathology , Mutation
Chinese Journal of Cardiology ; (12): 361-368, 2022.
Article in Chinese | WPRIM | ID: wpr-935155


Objective: To investigate the representability and etiological diagnostic value of myocardium samples obtained from patients with hypertrophic cardiomyopathy (HCM) by transthoracic echocardiography-guided percutaneous intramyocardial septal biopsy (myocardial biopsy of Liwen procedure). Methods: This study was a retrospective case-series analysis. Patients with HCM, who underwent myocardial biopsy of Liwen procedure and radiofrequency ablation in Xijing Hospital, Air Force Military Medical University from July to December 2019, were included. Demographic data (age, sex), echocardiographic data and complications were collected through electronic medical record system. The histological and echocardiographic features, pathological characteristics of the biopsied myocardium of the patients were analyzed. Results: A total of 21 patients (aged (51.2±14.5) years and 13 males (61.9%)) were enrolled. The thickness of ventricular septum was (23.3±4.5)mm and the left ventricular outflow tract gradient was (78.8±42.6)mmHg (1 mmHg=0.133 kPa). Eight patients (38.1%) were complicated with hypertension, 1 patient (4.8%) had diabetes, and 2 patients (9.5%) had atrial fibrillation. Hematoxylin-eosin staining of myocardial samples of HCM patients before radiofrequency ablation evidenced myocytes hypertrophy, myocytes disarray, nuclear hyperchromatism, hypertrophy, atypia, coronary microvessel abnormalities, adipocyte infiltration, inflammatory cell infiltration, cytoplasmic vacuoles, lipofuscin deposition. Interstitial fibrosis and replacement fibrosis were detected in Masson stained biopsy samples. Hematoxylin-eosin staining of myocardial samples of HCM patients after radiofrequency ablation showed significantly reduced myocytes, cracked nuclear in myocytes, coagulative necrosis, border disappearance and nuclear fragmentation. Quantitative analysis of myocardial specimens of HCM patients before radiofrequency ablation showed that there were 9 cases (42.9%) with mild myocardial hypertrophy and 12 cases (57.1%) with severe myocardial hypertrophy. Mild, moderate and severe fibrosis were 5 (23.8%), 9 (42.9%) and 7 (33.3%), respectively. Six cases (28.6%) had myocytes disarray. There were 11 cases (52.4%) of coronary microvessel abnormalities, 4 cases (19.0%) of adipocyte infiltration, 2 cases (9.5%) of inflammatory cell infiltration,6 cases (28.5%) of cytoplasmic vacuole, 16 cases (76.2%) of lipofuscin deposition. The diameter of cardiac myocytes was (25.2±2.8)μm, and the percentage of collagen fiber area was 5.2%(3.0%, 14.6%). One patient had severe replacement fibrosis in the myocardium, with a fibrotic area of 67.0%. The rest of the patients had interstitial fibrosis. The myocardial specimens of 13 patients were examined by transmission electron microscopy. All showed increased myofibrils, and 9 cases had disorder of myofibrils. All patients had irregular shape of myocardial nucleus, partial depression, mild mitochondrial swelling, fracture and reduction of mitochondrial crest, and local aggregation of myofibrillary interfascicles. One patient had hypertrophy of cardiomyocytes, but the arrangement of muscle fibers was roughly normal. There were vacuoles in the cytoplasm, and Periodic acid-Schiff staining was positive. Transmission electron microscopy showed large range of glycogen deposition in the cytoplasm, with occasional double membrane surround, which was highly indicative of glycogen storage disease. No deposition of glycolipid substance in lysozyme was observed under transmission electron microscope in all myocardial specimens, which could basically eliminate Fabry disease. No apple green substance was found under polarized light after Congo red staining, which could basically exclude cardiac amyloidosis. Conclusion: Myocardium biopsied samples obtained by Liwen procedure of HCM patients are representative and helpful for the etiological diagnosis of HCM.

Humans , Male , Biopsy/adverse effects , Cardiomegaly/pathology , Cardiomyopathy, Hypertrophic/diagnosis , Eosine Yellowish-(YS) , Fibrosis , Heart Defects, Congenital , Hematoxylin , Lipofuscin , Myocardium/pathology , Retrospective Studies
Chinese Journal of Burns ; (6): 616-628, 2022.
Article in Chinese | WPRIM | ID: wpr-940978


Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.

Animals , Female , Humans , Mice , Anhydrides , Endothelial Cells , Eosine Yellowish-(YS) , Gelatin/pharmacology , Graphite , Hematoxylin , Hydrogels/pharmacology , Methacrylates , Mice, Inbred C57BL , Neovascularization, Pathologic , Skin Abnormalities , Vascular Endothelial Growth Factor A
Rev. cuba. estomatol ; 58(4)dic. 2021.
Article in Spanish | LILACS-Express | LILACS, CUMED | ID: biblio-1408353


Introducción: Las técnicas de coloración histológica son útiles en el análisis ultraestructural de muestras tisulares, incluyendo el tejido gingival. Objetivo: Comparar la utilidad de tres métodos histoquímicos (hematoxilina-eosina, Masson Goldner y rojo sirio) en la identificación de elementos celulares y otros constituyentes del tejido gingival. Métodos: Estudio experimental in vitro que comprendió el análisis de tejidos gingivales de donantes sanos sin signos de inflamación gingival y con indicación de cirugía periodontal. Las muestras de encía se obtuvieron mediante gingivectomía, se procesaron e incluyeron en parafina, posteriormente se realizaron cortes con un micrótomo y se depositaron en portaobjetos de adhesión con polisina. Las muestras se agruparon y fueron teñidas con hematoxilina-eosina, Masson Goldner y rojo sirio, finalmente fueron visualizadas en un microscopio óptico Leica DM 750®. La lectura de los hallazgos fue realizada por patólogos orales. Resultados: La coloración hematoxilina-eosina evidencia elementos celulares y extracelulares del tejido epitelial y conectivo. Núcleos de color azul violeta, citoplasmas rosados, fibras de colágeno de matiz rosa claro, arteriolas y vénulas con túnica adventicia, media e íntima diferenciadas. La coloración Masson Goldner diferencia núcleos de coloración púrpura y citoplasma fucsia, presenta especificidad en identificar fibras de colágeno con tonalidad verde, distribuidas densa, homogénea y paralelamente en el tejido conectivo gingival. La tinción rojo sirio, permitió identificar las fibras de colágeno de color rosa brillante, mientras que el tejido epitelial y los vasos sanguíneos fueron de color amarillo. Conclusión: Cada coloración histológica evaluada en el presente trabajo tiene cierta afinidad y sensibilidad por estructuras celulares y componentes de la matriz extracelular específica. Su empleo es útil en el estudio de tejidos gingivales y podrían contribuir en el análisis de biopsias gingivales(AU)

Introduction: Histological staining techniques are useful in the ultrastructural analysis of tissue samples, including gingival tissue. Objective: Compare the usefulness of three histochemical methods (hematoxylin-eosin, Masson-Goldner and sirius red) for identification of cellular elements and other constituents of gingival tissue. Methods: An in vitro experimental study was conducted which included the analysis of gingival tissue from healthy donors without gingival inflammation signs and indication of periodontal surgery. The gum samples were obtained by gingivectomy, processed with paraffin, cut with a microtome and placed on Polysine adhesion slides. The samples were grouped, stained with hematoxylin-eosin, Masson Goldner and sirius red, and visualized under a Leica DM 750® microscope. Reading of the findings was performed by oral pathologists. Results: Hematoxylin-eosin staining found cellular and extracellular elements of the epithelial and connective tissue: violet-blue nuclei, pink cytoplasms, light rose collagen fibers, and arterioles and venules with differentiated tunica adventitia, media and intima. Masson-Goldner staining differentiated purple nuclei and fuchsia cytoplasm. It displayed specificity identifying green collagen fibers with dense, homogeneous and parallel distribution in the gingival connective tissue. Sirius red staining allowed identification of bright rose collagen fibers, whereas epithelial tissue and blood vessels were yellow. Conclusion: Each of the histological staining methods evaluated in the study shows a certain affinity with and sensitivity to cellular structures and components of the specific extracellular matrix. All three are useful for the study of gingival tissue and could contribute to the analysis of gingival biopsies(AU)

Humans , Connective Tissue/injuries , Gingivectomy , Hematoxylin , In Vitro Techniques , Collagen
Arq. bras. oftalmol ; 84(3): 271-274, May-June 2021. graf
Article in English | LILACS | ID: biblio-1248964


ABSTRACT This study aimed to analyze the anterior lens capsule specimens from both eyes of a patient with systemic sclerosis and compare them to the eyes of a control patient. No significant differences between systemic sclerosis and control eyes were observed in the results from the hematoxylin-eosin and picrosirius staining. In the samples obtained from both systemic sclerosis and control eyes, there were expressions of caspase, a molecule expressed in cell death by apoptosis. Heparanase was overexpressed in the systemic sclerosis sample compared to the control sample. Therefore, the anterior lens capsule of the patient with systemic sclerosis is probably affected by the disease since it showed marked expression of heparanase 1.(AU)

RESUMO Analisamos as amostras das cápsulas anteriores do cristalino de uma paciente com esclerose sistêmica e comparamos com as de um paciente controle. Não foram observadas diferenças significativas entre esclerose sistêmica e controle nos resultados da coloração com hematoxilina-eosina e picrosirius. Nas amostras obtidas da esclerose sistêmica e do controle, obtivemos expressão de caspase, uma molécula expressa na morte celular por apoptose. A heparinase foi expressa de forma mais marcante na amostra de esclerose sistêmica quando comparada ao controle. Portanto, a cápsula anterior do cristalino da paciente com esclerose sistêmica provavelmente foi afetada pela doença, uma vez que mostrou expressão aumentada de heparinase 1.(AU)

Humans , Scleroderma, Systemic/physiopathology , Heparin Lyase/administration & dosage , Hematoxylin , Lens Capsule, Crystalline/anatomy & histology
Pesqui. vet. bras ; 41: e06927, 2021. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1340346


Histopathology is an old science that is still currently utilized for disease diagnosis and research. The routinely processed histologic slides stained with hematoxylin and eosin are still used worldwide in most if not every histopathology laboratory. The technique is inexpensive, quick to perform, and allows the diagnosis of a fantastic variety of tissue changes and diseases. Skills in description and interpretation in histopathology are a craft that can be learned by repeatedly and systematically observing simple rules. In this article, we offer a few advices to help trainees in veterinary pathology at the start of their careers. Those advices are drawn from our experience in the diagnostic pathology routine and from the veterinary pathology literature. To enhance the understanding of these important steps in the histopathologic description of tissues, we decided to illustrate most concepts expressed here. We hope that our effort can add a bit to the development of future pathologists. Just like Alice, let us follow the White Rabbit into his burrow for this challenging experience!(AU)

A histopatologia é uma ciência antiga, mas ainda usada para diagnosticar e investigar a patogênese de doenças. As lâminas histológicas processadas rotineiramente e coradas por hematoxilina e eosina ainda são utilizadas em virtualmente todos os laboratórios de histopatologia do mundo. A técnica não é cara, é de execução rápida e permite o diagnóstico de uma fantástica variedade de doenças. As habilidades em descrever e interpretar os achados histopatológicos é um ofício que pode ser aprendido pela observação repetida e sistemática de regras simples. Para ajudar os estudantes de patologia no início de suas carreiras, abordamos aqui algumas dessas regras, extraídas tanto de nossa experiência quanto da literatura relacionada à patologia veterinária. Para aumentar a compreensão dos tópicos, decidimos ilustrar praticamente todos os conceitos expressados neste manuscrito. Esperamos que nosso esforço possa contribuir um pouco para o desenvolvimento de aspirantes a patologistas. Assim como Alice no País das Maravilhas, vamos seguir o coelho branco até a sua toca para essa aventura desafiadora.(AU)

Animals , Rabbits , Rabbits/anatomy & histology , Pathogenesis, Homeopathic , Hematoxylin
Rev. bras. ortop ; 55(6): 778-782, Nov.-Dec. 2020. graf
Article in English | LILACS | ID: biblio-1156183


Abstract Objectives The present paper aims to evaluate and compare the histological features of fresh and frozen menisci stored in a tissue bank for 1 month and for 5 years. Methods The meniscal grafts were subjected to a histological study. A total of 10 menisci were evaluated; 2 were frozen for 5 years, 4 were frozen for 1 month, and 4 were fresh, recently harvested specimens. Histological properties were evaluated in sections stained with hematoxylin and eosin and Masson trichrome methods. Results The menisci frozen for 1 month showed partially preserved collagen fiber structure and no significant hydropic tissue degeneration. The menisci frozen for 5 years presented an evident dissociation of collagen fibers and multiple foci of hydropic degeneration. Discussion Degeneration was much more significant in menisci stored for 5 years, indicating that a long freezing period results in substantial progression of tissue deterioration. This may suggest that the 5-year period, considered the maximum time for graft storage before transplant, is too long. Conclusion Grafts stored for 1 month showed a slight degenerative change in collagen fibers, whereas menisci frozen for 5 years presented significant tissue degeneration.

Resumo Objetivos Avaliar e comparar as características histológicas de meniscos frescos e meniscos congelados armazenados em banco de tecidos por 1 mês e por 5 anos. Métodos Foi feito um estudo histológico com enxertos meniscais. Avaliamos 10 meniscos, sendo 2 que ficaram armazenados sob congelamento por 5 anos, 4 armazenados congelados por 1 mês, e 4 frescos, recém captados. Foram feitos cortes histológicos corados com hematoxilina e eosina e Tricrômico de Masson, para avaliação das propriedades histológicas. Resultados Os meniscos congelados por 1 mês apresentaram preservação parcial da estrutura das fibras colágenas, sem degeneração hidrópica significativa do tecido. Nos meniscos congelados por 5 anos, observamos dissociação evidente das fibras colágenas, com presença de múltiplos focos de degeneração hidrópica. Discussão Encontramos degeneração bem mais significativa nos meniscos armazenados por 5 anos, o que indica que o longo período de congelamento leva à progressão significativa da degeneração do tecido. Isto pode sugerir que o período de 5 anos, considerado período máximo que o enxerto pode permanecer armazenado antes de ser transplantado, é um período muito longo. Conclusão Nos enxertos armazenados por 1 mês, existiu apenas discreta alteração degenerativa das fibras colágenas, enquanto que nos meniscos com 5 anos de congelamento foi observada degeneração significativa do tecido. Tibiais

Tissue Banks , Wounds, Penetrating , Collagen , Eosine Yellowish-(YS) , Transplants , Meniscus , Freezing , Goals , Hematoxylin
Rev. med. vet. zoot ; 67(3): 239-252, sep.-dic. 2020. graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1251919


RESUMEN Este trabajo describe los efectos del probiótico Saccharomyces cerevisiae sobre el área, número de criptas de Lieberkühn en duodeno y yeyuno, y producción de moco en ambas secciones intestinales de pollos de engorde. Fueron empleados los tejidos de un total de 27 individuos clasificados en un grupo control GC (n=12) y un grupo suplementado con probióticos GP (n=15). Los resultados revelaron que los grupos suplementados con el S. cerevisiae presentaron una mayor amplitud del área de las criptas en duodeno (p= 0,0119) y yeyuno (p= 0,0355), menor número de criptas por milímetro en duodeno (p= 0,0420) y mayor producción de moco en duodeno respecto al grupo control (p= 0,0185), mientras que en yeyuno no se observaron diferencias significativas. Se concluyó que el uso de Saccharomyces cerevisiae aumentó el tamaño del área de las criptas en ambas secciones intestinales y aumentó la producción de moco en duodeno; lo cual, al aumentar la superficie de absorción intestinal, seguramente podría resultar en mejoras de los parámetros productivos.

ABSTRACT This work describes the effects of the probiotic Saccharomyces cerevisiae on the area, number of Lieberkühn crypts in the duodenum and jejunum, and mucus production in both intestinal sections. Tissues from a total of 27 individuals were used, classified in control group - GC (n = 12) and group supplemented with probiotics - GP (n = 15). The results revealed that the group supplemented with S. cerevisiae presented a greater area of the crypts in the duodenum (p = 0.0119) and jejunum (p = 0.0355), less number of crypts per millimeter in the duodenum (p = 0.0420) and higher mucus production in the duodenum compared to the control group (p = 0.0185), while in the jejunum no significant differences were observed. It was concluded that the use of Saccharomyces cerevisiae increased the size of the crypt area in both intestinal sections and increased mucus production in the duodenum; which by increasing the intestinal absorption surface could surely result in improvements in the productive parameters.

Saccharomyces , Tissues , Chickens , Probiotics , Intestine, Small , Periodic Acid-Schiff Reaction , Eosine Yellowish-(YS) , Duodenum , Photograph , Hematoxylin , Intestinal Absorption
Rev. cuba. invest. bioméd ; 39(3): e640, jul.-set. 2020. tab, graf
Article in Spanish | LILACS, CUMED | ID: biblio-1138936


Introducción: La microscopía holográfica digital ha permitido a la microscopía óptica hacer uso de herramientas numéricas y computacionales; y esto, a su vez, ha favorecido múltiples avances en el estudio de las células y los tejidos en diferentes campos de la medicina y otras ciencias afines. Objetivo: Describir las características histológicas y morfométricas de los folículos tiroideos humanos con la microscopía holográfica digital. Métodos: Se realizó, desde el punto de vista histomorfométrico, un estudio descriptivo y transversal de folículos tiroideos humanos utilizando una instalación de microscopía holográfica digital. Se empleó la técnica de inclusión en parafina y tinción de hematoxilina-eosina para el procesamiento de las muestras. Se realizaron de 10 a 12 capturas de hologramas por muestra y el método de doble propagación para la reconstrucción de los hologramas. Se calculó el área, el perímetro, el diámetro mayor y menor de los folículos y cavidades foliculares y se realizaron reconstrucciones de imágenes holográficas en tres dimensiones. Se determinó como medida de tendencia central la media aritmética y como medida de dispersión la desviación típica o estándar. Resultados: Parámetros foliculares: área (5140,31 ± 1126,71 µm2); perímetro (2961,54 ± 71,2 µm); diámetro mayor:(921,17 ± 24,34 µm); diámetro menor: (746,67 ± 18,08 µm); altura del epitelio (7,92 ± 0,96). Cavidades foliculares: área (3686,18 ±1023,52 µm2); diámetro mayor: (698,86 ± 19,55 µm) y diámetro menor: (581,15 ± 13,82 µm). Conclusiones: Existen parámetros foliculares, determinados mediante la microscopía holográfica digital, no reportados por la literatura consultada, que resultan de interés en el estudio histológico de los folículos tiroideos humanos(AU)

Introduction: Digital holographic microscopy has made it possible to incorporate the use of numerical and computer tools into optical microscopy. This in turn has led to great progress in the study of cells and tissues in several fields of medicine and related sciences. Objective: Describe the histological and morphometric characteristics of human thyroid follicles using digital holographic microscopy. Methods: A descriptive cross-sectional histomorphometric study was conducted of human thyroid follicles using a digital holographic microscopy facility. Sample processing was based on inclusion technique by paraffin and hematoxylin-eosin staining. Ten to twelve holographic captures were made per sample, and the double propagation method was used for holographic reconstruction. Estimation was carried out of the area, perimeter, and greatest and smallest diameter of follicles and follicular cavities, and tri-dimensional reconstructions were made of holographic images. Arithmetic mean was determined as the measure of central tendency, and typical or standard deviation as the measure of dispersion. Results: Follicular parameters: area (5 140.31 ± 1 126.71 µm2); perimeter (2 961.54 ± 71.2 µm); greatest diameter (921.17 ± 24.34 µm); smallest diameter (746.67 ± 18.08 µm); epithelial height (7.92 ± 0.96). Follicular cavities: area (3 686.18 ± 1 023.52 µm2); greatest diameter (698.86 ± 19.55 µm); smallest diameter (581.15 ± 13.82 µm). Conclusions: A number of follicular parameters determined by digital holographic microscopy have not been reported by the literature consulted, and they are of interest to the histological study of human thyroid follicles(AU)

Humans , Computers , Holography/methods , Hematoxylin/therapeutic use , Thyroid Gland/physiology , Eosine Yellowish-(YS)
Acta odontol. Colomb. (En linea) ; 10(1): 24-36, 2020. ilus, ilus, graf, tab, ilus
Article in Spanish | LILACS, COLNAL | ID: biblio-1123490


Objetivo: establecer el tipo de patología más frecuente asociada a terceros molares incluidos extraídos en las clínicas de cirugía oral de la Facultad de Odontología de la Universidad Nacional de Colombia y la frecuencia de aparición de quistes dentígeros, rango de edad, sexo y la región anatómica en que predominan. Métodos: se revisaron 17 sacos foliculares de 11 pacientes atendidos en las clínicas de cirugía oral, entre agos-to del 2018 y febrero de 2019. Resultados: se recolectaron 7 biopsias de 5 pacientes masculinos y 10 de 7 pacientes femeninos, con edades comprendidas entre los 17 a 24 años (media: 20.40), se realizó estudio histopatológico con hematoxilina-eosina, cuyos datos obtenidos se analizaron para determinar frecuencia, edad, sexo y patología aso-ciada. Se encontraron 15 casos que mostraron cambios quísticos diagnosticados como quistes dentígeros y 2 casos diagnosticados como saco folicular. Conclusiones: aunque la muestra es pequeña, este estudio da indicios que los sacos foliculares asociados a terceros molares incluídos están altamente implicados en la formación de quistes dentígeros.

Objective: To establish the most frequent type of pathology associated to retained third molars extracted in the oral surgery clinics of the Dentistry School at Universidad Nacional de Colombia and the frequency of appearance of dentigerous cysts, age, sex and the anatomical region in which they predominate. Methods: A series of 17 follicular sacs were evaluated out of a total of 11 patients attended in the oral surgery clinics between August 2018 and February 2019. Results: Seven biopsies were collected from 5 male patients and ten from seven female patients aged between 17 and 24 years (mean: 20.40), histopathological study was performed with hematoxylin-eosin, the data obtained was analyzed to determine frequency, age, sex and associated pathology. It where found 15 cases that showed cystic changes diagnosed as dentigerous cysts and two cases diagnosed as follicular sac. Conclusions: Although the sample is small, this study gives indications that follicular sacs associated with retained third molars may be highly involved in the formation of dentigerous cysts.

Humans , Tooth, Impacted , Dentigerous Cyst , Pathology , Follicular Cyst , Diagnosis , Hematoxylin , Molar, Third
Rev. colomb. gastroenterol ; 35(1): 1-7, 2020. graf
Article in Spanish | LILACS | ID: biblio-1115595


Resumen Las técnicas empleadas para la detección del Helicobacter pylori (H. pylori) son no invasivas e invasivas. En estas últimas, la presencia del H. pylori se determina a partir de la tinción de hematoxilina-eosina (HE), prueba rutinaria, mientras que en pocas ocasiones se aplica la tinción de Warthin-Starry (WS) como coloración especial. Objetivo: identificar la presencia de H. pylori por medio de la coloración especial de la WS en biopsias de pacientes con gastritis crónica folicular, previamente negativas en la tinción HE. Materiales y métodos: se desarrolló un estudio de tipo descriptivo transversal, en un período de 12 meses. Se tomaron los bloques de parafina de las muestras de la mucosa gástrica de pacientes con diagnóstico de gastritis crónica e hiperplasia folicular. Además, se extrajo un corte histológico del mismo bloque, al cual se le aplicó HE y se determinó la presencia o ausencia de H. pylori. Así, de estar ausente, se tomó del mismo bloque un corte adicional y se aplicó WS. Esto se evaluó con el fin de identificar la existencia o no del bacilo. Resultados: se recolectaron 314 muestras; 209 fueron negativas y 105 fueron positivas para HE. El 45 % (94) de estas muestras fueron positivas respecto a la presencia del bacilo, al aplicar la segunda coloración, y el 55 % (115) de las muestras persistieron negativas. Conclusión: el hallazgo de H. pylori es significativamente alto al aplicar la coloración de WS a muestras cuyo estudio histológico evidenció la ausencia del bacilo en biopsias de la mucosa gástrica, especialmente en muestras con escasa cantidad de bacterias.

Abstract Non-invasive and invasive techniques can be used for detection of Helicobacter pylori. An invasive technique identifies the bacteria through routine hematoxylin-eosin staining. Warthin-Starry stain is rarely used. Objective: Our objective was to identify H. pylori by Warthin-Starry staining of patient's biopsies with chronic follicular gastritis who had previously tested negative in hematoxylin-eosin staining. Materials and methods: This is a descriptive, cross-sectional descriptive study that was carried out over a period of 12 months. The study examined paraffin blocks of samples taken from the gastric mucosa of patients diagnosed with chronic gastritis and follicular hyperplasia. A histological section was extracted from a block and tested with hematoxylin-eosin staining for the presence or absence of H. pylori. If absent, an additional cut was taken from the same block and Warthin-Starry staining was used to retest for the presence of the bacteria. Results: Of the 314 samples collected, 209 tested negative, and 105 tested positive for H. pylori when hematoxylin-eosin staining was used. Of the 209 negative samples, 45% (94) tested positive when Warthin Starry stain was used, and 55% (115) still tested negative. Conclusion: Findings of H. pylori are significantly higher when Warthin Starry stain was used to test samples whose previous histological study had evidenced an absence of the bacillus, especially in samples with a small amount of bacteria.

Humans , Male , Female , Helicobacter pylori , Gastritis , Hematoxylin , Hyperplasia , Bacteria , Gastric Mucosa
Rio de Janeiro; s.n; 2020. 125 p. graf, ilus, tab.
Thesis in Portuguese | LILACS | ID: biblio-1425346


A nefropatia isquêmica é uma doença renal crônica provocada pela redução do fluxo sanguíneo renal que pode progredir para a doença renal terminal, cujo tratamentos disponíveis se baseiam em terapias substitutivas da função renal, como diálise ou transplante renal. No entanto, devido ao alto custo dos tratamentos e a carência de órgãos, se faz necessária a busca por novas terapias, como as células-tronco (CT). Apesar do potencial terapêutico das CT em doenças crônicas, não está claro se essas células mantêm seus efeitos benéficos em órgãos lesionados por tempo prolongado. O objetivo desse estudo foi avaliar os efeitos precoces e tardios do tratamento com células-tronco adiposas (CTA) sobre a morfologia e o status oxidativo em rins de ratos com nefropatia isquêmica. A isquemia renal foi induzida pelo modelo 2rins-1clip (2R1C) e, depois de um mês da clipagem da artéria renal, foram injetadas 106 células-tronco na região subscapsular do rim afetado. Após 15 e 30 dias da injeção das CTA, a morfologia renal foi verificada por meio da análise macroscópica, microscópica e ultraestrutural. Além disso, o status oxidativo foi avaliado no tecido renal através da mensuração da atividade das enzimas antioxidantes catalase e glutationa peroxidase; e de marcadores biológicos de dano oxidativo, como proteínas carboniladas, 3-Nitrotirosina e 4-Hidroxinonenal. Por imunoperoxidase foi possível localizar as células-tronco adiposas GFP+ foram rastreadas e encontradas tanto 15 dias, quanto 30 dias após a injeção na região subcapsular. A restauração da arquitetura renal foi evidenciada 15d após o uso das células, onde detectamos redução na deposição de fibras colágenas no parênquima renal, o que não foi observado 30d após o uso das células. Os resultados também foram confirmados através da análise da ultraestrutura renal que mostraram restauração da arquitetura renal no grupo de 15d, não evidenciada no grupo de 30d. Quanto a análise do status oxidativo, somente os animais com nefropatia isquêmica mais prolongada apresentaram estresse oxidativo com redução da atividade da enzima antioxidante catalase no tecido renal. Além disso, foi observado dano proteico e lipídico, sem melhora dessa condição nos animais 30d após o tratamento com as células-tronco. No modelo de nefropatia isquêmica avaliado, o tratamento com CTA mostrou benefícios na morfologia renal a curto prazo, mas não tardiamente, apesar da permanência dessas células no tecido. Acreditamos que o estresse oxidativo, evidenciado somente no tecido renal com isquemia mais prolongada, possa ter dificultado a ação das células-tronco, contribuindo para tais achados. Esses resultados abrem perspectivas para o aprofundamento do estudo quanto à caracterização dos mecanimos de ação das CTA nas respostas anti-fibrogênicas, assim como o estabelecimento do número, frequência, vias de administração e melhor momento para uso dessas células no tratamento de doenças renais crônicas.

Ischemic nephropathy is a chronic kidney disease caused by reduced kidney blood flow that can progress to end stage kidney disease, whose available treatments are based on kidney function replacement therapies, such as dialysis or kidney transplantation. However, due to the high cost of treatments and the lack of organs, it is necessary to search for new therapies, such as stem cells (SC). Despite the therapeutic potential of SC in chronic diseases, it is unclear whether these cells maintain their beneficial effects on injured organs for a long time. The aim of this study was to evaluate the early and late effects of adipose-derived stem cells (ADSC) treatment on the morphology and oxidative status in kidneys of rats with ischemic nephropathy. Renal ischemia was induced by the 2kidneys-1clip (2K1C) model and, after a month of clipping the renal artery, 106 stem cells were injected into the subscapsular region of the affected kidney. After 15 and 30 days of ADSC injection, renal morphology was verified by macroscopic, microscopic, and ultrastructural analysis. In addition, oxidative status was assessed in renal tissue by measuring the activity of the antioxidant enzymes catalase and glutathione peroxidase; and biological markers of oxidative damage, such as carbonylated proteins, 3-nitrotyrosine and 4-hydroxynonenal. By immunoperoxidase, it was possible to locate GFP + adipose-derived stem cells that were tracked and found both 15 days and 30 days after injection in the subcapsular region. The restoration of the renal architecture was evidenced 15d after the use of the cells, where we detected a reduction in the deposition of collagen fibers in the renal parenchyma, which was not observed 30d after the use of the cells. The results were also confirmed by analyzing the renal ultrastructure, which showed restoration of the renal architecture in the 15d group, not evidenced in the 30d group. Regarding the analysis of oxidative status, only animals with more prolonged ischemic nephropathy presented oxidative stress with reduced activity of the antioxidant enzyme catalase in renal tissue. In addition, protein and lipid damage was observed, with no improvement in this condition in the animals 30d after treatment with stem cells. In the evaluated ischemic nephropathy model, treatment with ADSC showed benefits in renal morphology in the short term, but not late, despite the permanence of these cells in the tissue. We believe that oxidative stress, evidenced only in renal tissue with more prolonged ischemia, may have hindered the action of stem cells, contributing to such findings. These results open perspectives for further study on the characterization of ADSC mechanisms of action in anti-fibrogenic responses, as well as the establishment of the number, frequency, routes of administration and the best time to use these cells in the treatment of chronic kidney diseases.

Rats , Mesenchymal Stem Cells , Kidney/physiopathology , Kidney Diseases/chemically induced , Periodic Acid-Schiff Reaction/methods , Biomarkers/analysis , Catalase/analysis , Fluorescent Antibody Technique/methods , Oxidative Stress , Early Diagnosis , Protein Carbonylation , Delayed Diagnosis , Flow Cytometry/instrumentation , Glutathione Peroxidase/analysis , Hematoxylin
Article in Chinese | WPRIM | ID: wpr-827542


OBJECTIVE@#To explore the treatment conditions of acid decalcified specimens and improve the poor quality of sections and unclear structure of hematoxylin-eosin (HE) staining caused by the change in pH in tooth and hard tissue after acid decalcification.@*METHODS@#A total of 20 cases of oral pathological specimens that contain hard tissues were decalcified and treated with routine treatment, concentrated ammonia water immersion treatment, and saturated lithium carbonate solution immersion treatment. The quality and HE staining effects of hard tissue sections treated with different methods were compared.@*RESULTS@#Compared with routine treatment, lithium carbonate saturated solution treatment showed complete sections. Hematoxylin is strongly stained, the nucleus is clear, and the cytoplasm is bright.@*CONCLUSIONS@#Soaking acid decalcified specimens in lithium carbonate saturated solution before embedding in dehydration can neutralize the acidic environment of the tissue. The quality of sections and HE staining effect are improved and are suitable for the pretreatment of acid decalcified tissue samples of oral pathology.

Eosine Yellowish-(YS) , Hematoxylin , Staining and Labeling , Tooth
Rev. bras. ortop ; 54(6): 692-696, Nov.-Dec. 2019. graf
Article in English | LILACS | ID: biblio-1057962


Abstract Objective The present work aimed to evaluate the systemic effect of H. speciosa latex on bone neoformation. Methods For this, the latex was collected and diluted to 3% and 50%. A total of 28 Wistar rats were submitted to surgery to create a 5 mm diameter defect in the parietal bone. This experiment was conducted in 2 different periods: 1 and 2. For each period, the rats were divided into 3 groups: Control Group, Latex3 Group, and Latex50 Group, which received, respectively, daily administrations of 0.5 mL of distilled water, latex to 3% and latex to 50% by gavage, orally. The rats of periods 1 and 2 were euthanized, respectively, 15 and 30 days after the surgery, and the calvaria was collected. The results were analyzed using the ANOVA and Tukey tests; the significance level was 0.05. Results We show that, in each analyzed period, the experimental groups had the same amount of newly formed bone in the calvaria defect. Conclusion We conclude that daily and oral administrations of H. speciosa latex to 3% and to 50% over a period of 15 and 30 days does not contribute to the increase of the area of the newly formed bone in the calvaria defect.

Resumo Objetivo Este trabalho objetivou avaliar o efeito sistêmico do látex de H. speciosas obre a neoformação óssea. Métodos Para isso, o látex foi coletado e diluído a 3% e a 50%. Um total de 28 ratos Wistar foi submetido a cirurgia para a criação de um defeito de 5 mm de diâmetro no osso parietal. Esse experimento foi conduzido em dois períodos distintos: 1 e 2. Para cada período, os ratos foram divididos em 3 grupos: Grupo Controle, Grupo Látex3 e Grupo Látex50 que receberam, respectivamente, administrações diárias de 0,5 mL de água destilada, látex a 3% e látex a 50% por gavagem, via oral. Os ratos dos períodos 1 e 2 foram eutanasiados, respectivamente, 15 e 30 dias após a cirurgia e a calvária foi coletada. Os resultados foram analisados utilizando os testes ANOVA e Tukey; o nível de significância estabelecido foi 0,05. Resultados Mostramos que, em cada período analisado, os grupos experimentais tiveram a mesma quantidade de osso neoformado no defeito da calvária. Conclusão Portanto, concluímos que administrações diárias e orais do látex de H. speciosa a 3% e a 50% durante um período de 15 e 30 dias não contribui para o aumento da área do osso neoformado no defeito da calvária.

Animals , Rats , Bone and Bones , Complementary Therapies , Administration, Oral , Apocynaceae , Hematoxylin , Histology , Latex
Araçatuba; s.n; 2019. 97 p. ilus, graf, tab.
Thesis in Portuguese | BBO, LILACS | ID: biblio-1392501


A utilização de biomateriais que visam devolver o volume ósseo perdido após a perda dentária tem se expandido. O osso é um tecido conjuntivo altamente especializado, possuindo dinâmica aposicional onde o equilíbrio entre neoformação e reabsorção envolve a interação de fatores endócrinos, parácrinos e autócrinos. Diversas associações de materiais diversos e substitutos ósseos têm sido estudadas, porém, o MTA (Agregado Trióxido Mineral) ainda carece de informações acerca da sua utilização como substituto ósseo ou em associação com demais substitutos. Assim, o objetivo deste estudo foi avaliar a influência da presença do MTA Angelus Branco® nas proprorções de 5%, 10% e 15% em associação com substituto ósseo de Hidroxiapatita e ß- Tricálcio Fosfato na citotoxicidade, resposta tecidual e reparo ósseo, em defeito crítico em calvária de ratos. Para tanto, utilizou-se cultura celular da SAOS-2 para avaliação citológica e ensaio MTT do contato direto e eluentes. Também foram utilizados 112 ratos machos Wistar distribuídos em 7 grupos e avaliados em 2 tempos (7 e 28 dias). Após eutanasiados foram submetidos à microtomografia e por coloração de hematoxilina e eosina para análise histológica e histomorfométrica. A análise da homocedasticidade foi realizada pelo teste Shapiro-Wilk, para distinção dos dados paramétricos e não paramétricos. Para análise dos dados paramétricos, foi realizada análise de variância One-Way (ANOVA One-Way) e realizado o pós-teste de Tukey para os parâmetros microtomográficos e para a citotoxicidade foi aplicado o teste de correção de Bonferroni. Para os dados não paramétricos utilizou-se o Kruskal-Wallis e Mann Whitney. Adotou-se o nível de significância de p <0,05. Os parâmetros volume ósseo (BV), porcentagem de volume ósseo (BV/TV), espessura do trabeculado ósseo (Tb.Th) e número de trabéculas (Tb.N) não apresentaram diferenças estatísticas. Sendo o grupo Osteosynt® +15% MTA o grupo com maiores valores médios para os parâmetros BV, BV/TV, Tb.Th e Tb.N. A análise histomorfométrica para neoformação óssea e reabsorção dos biomateriais não apresentaram diferenças estatísticas. De acordo com os resultados, pode-se concluir que, estatisticamente, a adição de MTA nas 3 proporções testadas não demonstrou efetividade quando comparada ao uso isolado dos substitutos ósseos Bio-Oss® e Osteosynt® e coágulo. No entanto, foi evidenciada tendências à superioridade numérica quanto ao volume ósseo neoformado da associação Osteosynt® + 15% MTA aos 7 dias e Osteosynt® + 5% MTA e Osteosynt® + 10% MTA nas análises aos 28 dias(AU)

The use of biomaterials that aim to return lost bone volume after tooth loss has expanded. Bone is a highly specialized connective tissue with appositional dynamics where the balance between neoformation and resorption involves the interaction of endocrine, paracrine and autocrine factors. Several associations of different materials and bone substitutes have been studied. However, the MTA (Mineral Trioxide Aggregate) still lacks information about its use as bone substitute or in association with other substitutes. Thus, the aim of this study was to evaluate the influence of presence of MTA Angelus Branco® in the 5%, 10% and 15% proportions in association with Hydroxyapatite and ß-Tricalcium Phosphate bone substitute in cytotoxicity, tissue response and bone repair in critical defect in rat calvaria. For this, SAOS-2 cell culture was used for cytological evaluation and MTT assay of direct contact and eluents. We also used 112 male Wistar rats distributed into 7 groups and evaluated at 2 distinct times (7 and 28 days). After euthanasia, the specimens were submitted to microtomography and hematoxylin and eosin staining for histological and histomorphometric analysis. The analysis of homoscedasticity was performed by the Shapiro-Wilk test for distinction of parametric and non-parametric data. For analysis of parametric data, one-way analysis of variance (One-Way ANOVA) was performed and Tukey's post-test was performed for the microtomographic parameters and for the cytotoxicity the Bonferroni correction test was applied. For non-parametric data we used the Kruskal-Wallis and Mann Whitney. The significance level of p <0.05 was adopted. The parameters bone volume (BV), bone volume percentage (BV / TV), bone trabecular thickness (Tb.Th) and number of trabeculae (Tb.N) showed no statistical differences. The Osteosynt® + 15% MTA group was the group with the highest mean values for the parameters BV, BV / TV, Tb.Th and Tb.N. Histomorphometric analysis for bone neoformation and biomaterial resorption showed no statistical differences. According to the results, it can be concluded that, statistically, the addition of MTA in the 3 tested proportions did not show effectiveness when compared to the isolated use of the Bio-Oss® and Osteosynt® bone substitutes and clot. However, trends in numerical superiority regarding the newly formed bone volume of the association Osteosynt® + 15% MTA at 7 days and Osteosynt® + 5% MTA and Osteosynt® + 10% MTA in the analyzes at 28 days were evidenced(AU)

Animals , Rats , Bone Regeneration , Bone Substitutes , Biocompatible Materials , Bone and Bones , Cell Survival , Tooth Loss , Rats, Wistar , Durapatite , Hematoxylin
Article in English | WPRIM | ID: wpr-764061


BACKGROUND AND OBJECTIVE: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. METHODS AND RESULTS: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza®) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. CONCLUSIONS: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.

Humans , Biology , Bone Marrow , Coloring Agents , Endothelial Cells , Eosine Yellowish-(YS) , Hematopoietic Stem Cells , Hematoxylin , Immunohistochemistry , Mesenchymal Stem Cells , Methods , Organoids , Regenerative Medicine , Stem Cells , Umbilical Cord , Vimentin
Article in English | WPRIM | ID: wpr-763356


OBJECTIVE: To compare black rice (Oryza sativa L) extract with three different staining methods for human sperm head assessment. METHODS: Semen samples were collected from 34 volunteers. Four smears of each ejaculate were prepared for staining using the rapid Papanicolaou (PAP) stain, SpermBlue, DipQuick, and black rice extract. The percentage of defective sperm heads (mean±standard deviation) was compared. RESULTS: Black glutinous rice extract, a natural dye, was used instead of hematoxylin to stain the nuclei of the sperm heads. The percentage of defective sperm heads showed a significant difference between black rice extract and DipQuick (p=0.000). In contrast, black rice extract and rapid PAP showed no statistically significant difference (p=0.974). A strong correlation (r =0.761) was found between the findings obtained using rapid PAP and black rice extract. In contrast, a weak correlation (r =0.248) was obtained between DipQuick and black rice extract for the percentage of defective sperm heads. CONCLUSION: The results showed good agreement and a strong correlation between the rapid PAP and black rice extract stains. The advantages of black rice extract as a novel substitute for hematoxylin for nuclear staining include ease of preparation, local availability, and favorable nuclear staining properties. Further studies could also focus on comparing staining techniques in clinical samples.

Humans , Coloring Agents , Hematoxylin , Semen , Sperm Head , Spermatozoa , Volunteers
Article in English | WPRIM | ID: wpr-761910


BACKGROUND: Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation in vitro and bone regeneration in vivo. METHODS: The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on in vivo bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining. RESULTS: KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10 µM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model. CONCLUSION: These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.

Animals , Mice , Alkaline Phosphatase , Bone Regeneration , Collagen , Eosine Yellowish-(YS) , Hematoxylin , In Vitro Techniques , Miners , Osteoblasts , Osteogenesis , Porifera , Real-Time Polymerase Chain Reaction , Xanthine
Article in English | WPRIM | ID: wpr-785606


OBJECTIVE: Sequelae of behavioral impairments associated with human traumatic brain injury (TBI) include neurobehavioral problems. We compared exploratory, cognitive, and depressive-like behaviors in pediatric and adult male mice exposed to controlled cortical impact (CCI).METHODS: Pediatric (21 to 25 days old) and adult (8 to 12 weeks old) male C57Bl/6 mice underwent CCI at a 2-mm depth of deflection. Hematoxylin and eosin staining was performed 3 to 7 days after recovery from CCI, and injury volume was analyzed using ImageJ. Neurobehavioral characterization after CCI was performed using the Barnes maze test (BMT), passive avoidance test, open-field test, light/dark test, tail suspension test, and rotarod test. Acutely and subacutely (3 and 7 days after CCI, respectively), CCI mice showed graded injury compared to sham mice for all analyzed deflection depths.RESULTS: Time-dependent differences in injury volume were noted between 3 and 7 days following 2-mm TBI in adult mice. In the BMT, 2-mm TBI adults showed spatial memory deficits compared to sham adults (P < 0.05). However, no difference in spatial learning and memory was found between sham and 2-mm CCI groups among pediatric mice. The open-field test, light/dark test, and tail suspension test did not reveal differences in anxiety-like behaviors in both age groups.CONCLUSION: Our findings revealed a graded injury response in both age groups. The BMT was an efficient cognitive test for assessing spatial/non-spatial learning following CCI in adult mice; however, spatial learning impairments in pediatric mice could not be assessed.

Animals , Humans , Male , Mice , Adult , Brain Injuries , Eosine Yellowish-(YS) , Hematoxylin , Hindlimb Suspension , Learning , Memory , Rotarod Performance Test , Spatial Learning , Spatial Memory