RESUMO
Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.
Assuntos
Intestinos , Traumatismo por Reperfusão , Camundongos , Animais , Histona-Lisina N-Metiltransferase/metabolismo , Traumatismo por Reperfusão/metabolismo , Células Epiteliais/metabolismo , Isquemia/metabolismoRESUMO
Recent physiological studies have shown that the deep fascia has received much attention concerning clinical medicine; however, histological examination of the deep fascia has not been well established. In this study, we aimed to clarify and visualize the structure of the deep fascia by taking advantage of cryofixation techniques and low-vacuum scanning electron microscopy. As a result, the ultrastructural observations revealed three-dimensional stratification of the deep fascia composed of three layers: the first superficial layer consisting of collagen fibers extending in various directions with blood vessels and peripheral nerves; the second intermediate layer formed by single straight and thick collagen fibers with flexibility; and the third deepest layer, consisting of relatively straight and thin collagen fibers. We explored the use of two hooks to hold a piece of deep fascia in place through the course of cryo-fixation. A comparative observation with or without the hook-holding procedure would indicate the morphological adaptation to physiological stretch and contraction of the deep fascia. The present morphological approach paves the way to visualize three-dimensional ultrastructures for future biomedical studies including clinical pathophysiology.
Assuntos
Colágeno , Fáscia , Fáscia/fisiologia , Vácuo , Microscopia Eletrônica de Varredura , Colágeno/ultraestrutura , Microscopia ConfocalRESUMO
ABSTRACT: In the putrefied brain, the cortex and basal ganglia show dark-grayish to green discoloration due to sulfhemoglobin formed from hydrogen sulfide (H 2 S) produced by endogenous bacteria and hemoglobin. In this study, we propose and demonstrate another mechanism of green discoloration in the brain. The formalin-fixed brain of a cadaver donated for medical education with no putrefaction was used. Half of the brain was immersed in sodium hydrosulfide solution, to imitate the H 2 S produced by bacteria. This half showed greenish discoloration, mainly in the basal ganglia and cortex. The other half showed positive Perls' Prussian blue staining, mainly in the basal ganglia and cortex. The area of greenish discoloration due to H 2 S and the region positive for Perls' Prussian blue staining coincided. Tissue treatment with strong oxidizing agents is required to liberate heme iron. The positive Perls' Prussian blue staining in this study thus does not reflect heme iron. In conclusion, we considered that non-heme iron compounds physiologically present in the brain and H 2 S represent sources of putrefactive greenish discoloration in the brain.
Assuntos
Sulfeto de Hidrogênio , Ferro , Humanos , Encéfalo , Coloração e Rotulagem , Mudanças Depois da MorteRESUMO
BACKGROUND: Clavicle fractures are common injuries, especially in young, active individuals. Operative treatment is recommended for completely displaced clavicle shaft fractures, and plate fixation is stronger than the use of intramedullary nails. Few studies have reported on iatrogenic injuries to the muscle attached to the clavicle during fracture surgery. The aim of this study was to clarify the area of the insertion sites of muscles attached to the clavicle in Japanese cadavers using gross anatomy and three-dimensional (3D) analysis. We also aimed to compare the effects of anterior plate templating and superior plate templating on clavicle shaft fractures using 3D images. METHODS: Thirty-eight clavicles from Japanese cadavers were analyzed. We removed all clavicles to identify the insertion sites and measured the size of the insertion area of each muscle. Three-dimensional templating was performed on both the superior and anterior plates of the clavicle using data obtained from computed tomography. The areas covered by these plates on the muscles attached to the clavicle were compared. Histological examination was performed on four randomly selected specimens. RESULTS: The sternocleidomastoid muscle was attached proximally and superiorly; the trapezius muscle was attached posteriorly and partly superiorly; and the pectoralis major muscle and deltoid muscles were attached anteriorly and partially superiorly. The non-attachment area was located mainly in the posterosuperior part of the clavicle. It was difficult to distinguish the borders of the periosteum and pectoralis major muscles. The anterior plate covered a significantly broader area (mean 6.94 ± 1.36 cm2) of the muscles attached to the clavicle than did the superior plate (mean 4.11 ± 1.52 cm2) (p < 0.0001). On microscopy, these muscles were inserted directly into the periosteum. CONCLUSION: Most of the pectoralis major and deltoid muscles were attached anteriorly. The non-attachment area was located mainly from the superior to posterior part of the clavicle midshaft. Both macroscopically and microscopically, the boundaries between the periosteum and these muscles were difficult to demarcate. The anterior plate covered a significantly broader area of the muscles attached to the clavicle than that by the superior plate.
Assuntos
Clavícula , Fraturas Ósseas , Humanos , Clavícula/diagnóstico por imagem , Clavícula/cirurgia , Músculos Peitorais , Periósteo , Placas Ósseas , Cadáver , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/cirurgiaRESUMO
Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, â¼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.
Assuntos
Antígenos de Plaquetas Humanas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Trombocitopenia , Animais , Humanos , Coelhos , Transfusão de Plaquetas/efeitos adversos , Células-Tronco Pluripotentes Induzidas/metabolismo , Plaquetas/metabolismo , Trombocitopenia/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversosRESUMO
Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Progenitoras de Megacariócitos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Plaquetas/metabolismo , Linhagem Celular , Proliferação de Células , Células Clonais , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima , Proteína bcl-X/metabolismoRESUMO
Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.
Assuntos
Ouro/análise , Nanopartículas Metálicas/análise , Animais , Feminino , Imuno-Histoquímica , Rim/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/análise , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Ovário/ultraestrutura , Coelhos , Ratos Wistar , Coloração e RotulagemRESUMO
Beginning of metastasis, cancer cells detach from the primary tumor and they can survive even under loss of anchorage; however, the detachment-elicited mechanisms have remained unknown. Here, we found that Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells is dynamically translocated from intracellular vesicles to the plasma membrane when the attached cells are detached and that this mechanism contributes to the survival of the detached (floating) cancer cells. α3NaK was detected in the plasma membrane of floating cancer cells in peritoneal fluids of patients, while it was in the cytoplasm of the cells in primary tumor tissues. On cancer cell detachment, we also found the focal-adhesion-kinase-dependent Ca2+ response that induces the α3NaK translocation via nicotinic acid adenine dinucleotide phosphate pathway. Activation of AMP-activated protein kinase was associated with the translocated α3NaK in the plasma membrane. Collectively, our study identifies a unique mechanism for survival of detached cancer cells, opening up new opportunities for development of cancer medicines.
RESUMO
Mini-abstract: Application of a three-dimensional culture system with air exposure facilitates the formation of large cell spheres possessing cribriform glands and producing mucin in the collagen gel. Transmission electron microscopy revealed the formation of microvilli and junctional complexes at the apical side of the cell. This study aimed to reproduce the characteristics of original adenocarcinoma tumors in vitro. The pancreatic cell line, SUIT-58, derived from a moderately differentiated adenocarcinoma of metastatic pancreatic cancer was used. The cells have a sheet structure in conventional cell culture without forming glands or exhibiting mucin production in the lumen. First, the necessity of scaffolds to create an adenocarcinoma-like microenvironment for SUIT-58 pancreatic cancer cells was assessed. Compared with conventional culture plates, the use of type I collagen as a scaffold played an important role in the formation of densely congested microvilli, as observed through scanning electron microscopy. As gland formation is one of the features of adenocarcinoma, we also assessed gland formation. Use of a recently developed three-dimensional culture system with air exposure resulted in the formation of large cell spheres possessing cribriform glands, which released mucin into the lumen. Transmission electron microscopy also revealed the formation of microvilli in the lumen of the glands and junctional complex at the intercellular part, which were similar to those observed in xenografts. These findings indicate that an in vitro three-dimensional culture system with air exposure reflects the intrinsic features of the original tumor, suggesting that this culture system could be useful for preliminary research of certain cancers.
Assuntos
Adenocarcinoma/ultraestrutura , Técnicas de Cultura de Células , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Neoplasias Pancreáticas/ultraestrutura , Adenocarcinoma/patologia , Humanos , Neoplasias Pancreáticas/patologia , Esferoides Celulares , Alicerces Teciduais , Células Tumorais Cultivadas , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation, progressive bone marrow (BM) fibrosis, splenomegaly, and anemia. BM fibrosis was previously thought to be a reactive phenomenon induced by mesenchymal stromal cells that are stimulated by the overproduction of cytokines such as transforming growth factor (TGF)-ß1. However, the involvement of neoplastic fibrocytes in BM fibrosis was recently reported. In this study, we showed that the vast majority of collagen- and fibronectin-producing cells in the BM and spleens of Jak2V617F-induced myelofibrosis (MF) mice were fibrocytes derived from neoplastic hematopoietic cells. Neoplastic monocyte depletion eliminated collagen- and fibronectin-producing fibrocytes in BM and spleen, and ameliorated most characteristic MF features in Jak2V617F transgenic mice, including BM fibrosis, anemia, and splenomegaly, while had little effect on the elevated numbers of megakaryocytes and stem cells in BM, and leukothrombocytosis in peripheral blood. TGF-ß1, which was produced by hematopoietic cells including fibrocytes, promoted the differentiation of neoplastic monocytes to fibrocytes, and elevated plasma TGF-ß1 levels were normalized by monocyte depletion. Collectively, our data suggest that neoplastic fibrocytes are the major contributor to BM fibrosis in PMF, and TGF-ß1 is required for their differentiation.
Assuntos
Fibroblastos/patologia , Janus Quinase 2/metabolismo , Megacariócitos/patologia , Mutação , Mielofibrose Primária/patologia , Animais , Diferenciação Celular , Proliferação de Células , Fibroblastos/metabolismo , Janus Quinase 2/genética , Megacariócitos/metabolismo , Camundongos , Camundongos Transgênicos , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo , Esplenomegalia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-I-deficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Diferenciação Celular , Antígenos de Histocompatibilidade Classe I/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Animais , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Técnicas de Inativação de Genes , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Microglobulina beta-2/deficiência , Microglobulina beta-2/genéticaRESUMO
The purpose of this study was to clarify the impact of self-exercise for elderly patients in an acute hospital after hip fracture. This retrospective observational study used data from the Japan Rehabilitation Database spanning 2005-2015. This study identified in-hospital hip fracture patients admitted to an acute hospital. After applying exclusion criteria, 375 patients were eligible. The primary outcome was motor Functional Independence Measure (FIM) efficiency. Of the patients with hip fracture, 39% performed self-exercises. Patients who performed self-exercise had significantly higher motor FIM efficiency than those who did not (1.22 vs. 0.79 ; P ?0.01). Multivariable regression analysis showed that motor FIM efficiency was significantly and positively correlated with self-exercise (coefficient, 0.25 ; 95% confidence interval, 0.13 to 0.43 ; P ?0.01). The data suggest that self-exercise is associated with good rehabilitation outcomes in hip fracture patients. J. Med. Invest. 66 : 178-181, February, 2019.
Assuntos
Exercício Físico , Fraturas do Quadril/reabilitação , Idoso , Idoso de 80 Anos ou mais , Feminino , Fraturas do Quadril/fisiopatologia , Humanos , Masculino , Estudos RetrospectivosRESUMO
BACKGROUND: Monocyte-derived fibrocytes play an important role in the progression of fibrosis in the skin, lungs, heart and kidney. However, the contribution of fibrocytes to liver fibrosis is unclear. The aim of this study was to investigate whether fibrocytes contributed to fibrosis progression in the livers of carbon tetrachloride (CCl4)-treated mice. METHODS: C57BL/6J mice were divided into 4 groups: normal control group, CCl4-treated group, CCl4â¯+â¯control liposome-treated group, and CCl4â¯+â¯clodronate liposome-treated group. For the elimination of systemic monocyte and monocyte-derived fibrocyte, one group was treated with clodronate liposome, and another group with control liposome as a control. After 4 weeks of treatment, hepatic mononuclear cells were subjected to immunofluorescent (IF) staining and fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes. Measurement of collagen-positive Sirius red stained area and collagen-I mRNA expression in the liver were performed to evaluate the degree of liver fibrosis quantitatively. RESULTS: In the liver of the CCl4-treated and CCl4â¯+â¯control liposome-treated groups, the number of fibrocytes, the area positive for Sirius red staining and collagen-I mRNA expression significantly increased compared with those in the normal control group. In the liver of the CCl4â¯+â¯clodronate liposome-treated group, few fibrocytes was observed as in the normal control group, but Sirius red staining positive area and collagen-I mRNA expression were increased and equivalent to the CCl4-treated and CCl4â¯+â¯control liposome-treated groups. CONCLUSION: Monocyte-derived fibrocytes play a minimal role in CCl4-induced liver fibrosis. Cells other than fibrocytes such as hepatic stellate cells play a central role in liver fibrosis.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Cirrose Hepática Experimental/patologia , Fígado/patologia , Monócitos/patologia , Animais , Tetracloreto de Carbono , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Ácido Clodrônico/farmacologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Progressão da Doença , Feminino , Fígado/efeitos dos fármacos , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Fatores de Tempo , Regulação para CimaRESUMO
During maturation, megakaryocytes (MKs) express ß1-tubulin (TUBB1) and rearrange their microtubule components to enlarge, form proplatelets, and eventually release platelets. The development of a platform to identify in vitro conditions that would efficiently promote MK development could potentially enable large-scale platelet production. Here, we show that an immortalized MK cell line (imMKCL) genetically modified to express the ß1-tubulin-Venus reporter provides a practical system to efficiently monitor the in vitro production of platelet-like particles (PLPs). The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9, and the expression was visualized by Venus fluorescence intensity. This imMKCL reporter line was then used for high-throughput drug screening. We identified several compounds that significantly improved the efficiency of PLP production in vitro under feeder-free conditions and showed a significant tendency to recover platelets in vivo in a mouse thrombocytopenia model induced by anti-GPIbα antibody administration. Interestingly, most of these compounds, including a WNT signaling pathway inhibitor, Wnt-C59, antagonized the aryl hydrocarbon receptor (AhR) to increase PLP production, confirming the crucial role of AhR inhibition in MK maturation. Consistently, small interfering RNA treatment against AhR increased the Venus intensity and PLP production. TCS 359, an FLT3 inhibitor, significantly increased PLP production independently of FLT3 or AhR. This study highlights the usefulness of the ß1-tubulin reporter MK line as a useful tool to study the mechanisms underlying thrombopoiesis and to identify novel inducers of ex vivo platelet production.
Assuntos
Plaquetas/citologia , Descoberta de Drogas/métodos , Genes Reporter/genética , Megacariócitos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Luciferases/genética , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/metabolismo , TrombopoeseRESUMO
The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.
Assuntos
Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Trombopoese/fisiologia , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Humanos , Hidrodinâmica , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Megacariócitos/fisiologiaRESUMO
Recent advances in bio-medical research, such as the production of regenerative organs from stem cells, require three-dimensional analysis of cell/tissue architectures. High-resolution imaging by electron microscopy is the best way to elucidate complex cell/tissue architectures, but the conventional method requires a skillful and time-consuming preparation. The present study developed a three-dimensional survey method for assessing cell/tissue architectures in 30-µm-thick paraffin sections by taking advantage of backscattered electron imaging in a low-vacuum scanning electron microscope. As a result, in the kidney, the podocytes and their processes were clearly observed to cover the glomerulus. The 30 µm thickness facilitated an investigation on face-side (instead of sectioned) images of the epithelium and endothelium, which are rarely seen within conventional thin sections. In the testis, differentiated spermatozoa were three-dimensionally assembled in the middle of the seminiferous tubule. Further application to vascular-injury thrombus formation revealed the distinctive networks of fibrin fibres and platelets, capturing the erythrocytes into the thrombus. The four-segmented BSE detector provided topographic bird's-eye images that allowed a three-dimensional understanding of the cell/tissue architectures at the electron-microscopic level. Here, we describe the precise procedures of this imaging method and provide representative electron micrographs of normal rat organs, experimental thrombus formation, and three-dimensionally cultured tumour cells.
Assuntos
Técnicas Histológicas/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Parafina/química , Animais , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Coelhos , Ratos , Ratos Wistar , Células Tumorais Cultivadas , VácuoRESUMO
BACKGROUND: Rehabilitation characteristics in high-performance hospitals after acute stroke are not clarified. This retrospective observational study aimed to clarify the characteristics of high-performance hospitals in acute stroke rehabilitation. METHODS: Patients with stroke discharged from participating acute hospitals were extracted from the Japan Rehabilitation Database for the period 2006-2015. We found 6855 patients from 14 acute hospitals who were eligible for analysis in this study after applying exclusion criteria. We divided facilities into high-performance hospitals and low-performance hospitals using the median of the Functional Independent Measure efficiency for each hospital. We compared rehabilitation characteristics between high- and low-performance hospitals. RESULTS: High-performance hospitals had significantly shorter length of stay. More patients were discharged to home in the high-performance hospitals compared with low-performance hospitals. Patients in high-performance hospitals received greater amounts of physical, occupational, and speech therapy. Patients in high-performance hospitals engaged in more self-exercise, weekend exercise, and exercise in wards. There was more participation of board-certified physiatrists and social workers in high-performance hospitals. CONCLUSIONS: Our data suggested that amount, timing, and type of rehabilitation, and participation of multidisciplinary staff are essential for high performance in acute stroke rehabilitation.
Assuntos
Hospitais , Qualidade da Assistência à Saúde , Reabilitação do Acidente Vascular Cerebral , Humanos , Tempo de Internação , Estudos Retrospectivos , Acidente Vascular Cerebral , Reabilitação do Acidente Vascular Cerebral/métodosRESUMO
Estrogen affects mitochondrial function in various tissues, but the precise mechanism remains unclear. We, therefore investigated the effect on estrogen-regulated mitochondrial morphology by dynamin-related protein 1 (Drp1) and its Ser616-phosphorylated derivative (pDrp1Ser616) are involved in mitochondrial fission. MCF7 human breast cancer cells were treated with 17ß-estradiol (E2), an estrogen receptor (ER) α and ß antagonist (ICI 182, 780), an ERα antagonist (MPP), and an ERß antagonist (PHTPP) for 24 hr. The expression of Drp1 and pDrp1Ser616 was analyzed by western blotting and immunohistochemistry. Mitochondrial morphology was analyzed by transmission electron microscopy (TEM). In control cells, Drp1 was detected in the cytoplasm of all cells while pDrp1 was observed in the cytoplasm of 3.4 ± 1.0% of the total population. After E2 treatment, pDrp1Ser616-positive cells comprised 30.6 ± 5.6% of the total population, 10.5 ± 1.7% after E2 + ICI treatment, 12.4 ± 4.2% after E2 + MPP treatment, and 24.0 ± 2.2% after E2 + PHTPP treatment. In ERα knockdown MCF7 cells, pDrp1 expression was decreased after E2 treatment compared to E2-treated wild type cells. Tubular pattern mitochondria were found in the control cells but the number of short and small pattern mitochondria (< 0.5 µm2) was significantly increased after E2 treatment (as observed by TEM). We, therefore concluded that the phosphorylation of Drp1 is important for E2-dependent mitochondrial morphological changes through ERα.