RESUMO
The correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1(cond) and Npn1(Sema-) to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre(+);Npn1(cond-/-) mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1(Sema-) mutants appeared anatomically normal. Furthermore, Olig2-Cre(+);Npn1(cond) mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1(Sema-) mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre(+);Npn1(cond-/-) mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1(Sema-) mutants to compensate for their developmental deficits.
Assuntos
Neurônios Motores/metabolismo , Neuropilina-1/metabolismo , Semaforina-3A/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Axônios/fisiologia , Axônios/ultraestrutura , Peso Corporal/genética , Peso Corporal/fisiologia , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Osso e Ossos/embriologia , Osso e Ossos/inervação , Osso e Ossos/metabolismo , Membro Anterior/embriologia , Membro Anterior/crescimento & desenvolvimento , Membro Anterior/inervação , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/inervação , Fibras Nervosas/metabolismo , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Neuropilina-1/genética , Semaforina-3A/genética , Transdução de Sinais/genética , Fatores de TempoRESUMO
The development of hybrid optical tomography methods to improve imaging performance has been suggested over a decade ago and has been experimentally demonstrated in animals and humans. Here we examined in vivo performance of a camera-based hybrid fluorescence molecular tomography (FMT) system for 360° imaging combined with X-ray computed tomography (XCT). Offering an accurately co-registered, information-rich hybrid data set, FMT-XCT has new imaging possibilities compared to stand-alone FMT and XCT. We applied FMT-XCT to a subcutaneous 4T1 tumor mouse model, an Aga2 osteogenesis imperfecta model and a Kras lung cancer mouse model, using XCT information during FMT inversion. We validated in vivo imaging results against post-mortem planar fluorescence images of cryoslices and histology data. Besides offering concurrent anatomical and functional information, FMT-XCT resulted in the most accurate FMT performance to date. These findings indicate that addition of FMT optics into the XCT gantry may be a potent upgrade for small-animal XCT systems.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Tomografia Óptica/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Remodelação Óssea , Modelos Animais de Doenças , Desenho de Equipamento , Feminino , Fluorescência , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/patologia , Camundongos , Osteogênese Imperfeita/diagnóstico por imagem , Osteogênese Imperfeita/patologiaRESUMO
Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients.
Assuntos
Sistema Cardiovascular/fisiopatologia , Colágeno Tipo I/genética , Pulmão/fisiopatologia , Osteogênese Imperfeita/fisiopatologia , Adolescente , Animais , Insuficiência da Valva Aórtica/fisiopatologia , Criança , Pré-Escolar , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos , Miocárdio/metabolismo , Osteogênese Imperfeita/genética , Fenótipo , Insuficiência da Valva Pulmonar/fisiopatologia , Escoliose/etiologia , Adulto JovemRESUMO
Within the Munich, Germany, N-ethyl-N-nitrosourea mouse mutagenesis program, we isolated a dominant Jak1 mouse model resembling phenotypic characteristics related to autoimmune disease. Chromosomal sequencing revealed a new Jak1 (p.Ser645Pro) point mutation at the conserved serine of the pseudokinase domain, corresponding to a somatic human mutation (p.Ser646Phe) inducing a constitutive activation of the Janus kinase (JAK)/STAT pathway. Morphologically, all Jak1(S645P+/-) mice showed a progressive structural deterioration of ears starting at the age of 4 months, with mononuclear cell infiltration into the dermis. Female mutant mice, in particular, developed severe skin lesions in the neck from 7 months of age. The IHC analysis of these lesions showed an activation of Stat3 downstream to Jak1(S645P) and elevated tissue levels of IL-6. Histopathological analysis of liver revealed a nodular regenerative hyperplasia. In the spleen, the number of Russell bodies was doubled, correlating with significant increased levels of all immunoglobulin isotypes and anti-DNA antibodies in serum. Older mutant mice developed thrombocytopenia and altered microcytic red blood cell counts. Jak1(S645P+/-) mice showed phenotypes related to impaired bone metabolism as increased carboxy-terminal collagen cross-link-1 levels and alkaline phosphatase activities in plasma, hypophosphatemia, and strongly decreased bone morphometric values. Taken together, Jak1(S645P+/-) mice showed an increased activation of the IL-6-JAK-STAT pathway leading to a systemic lupus erythematosus-like phenotype and offering a new valuable tool to study the role of the JAK/STAT pathway in disease development.
Assuntos
Doenças Autoimunes/genética , Janus Quinase 1/genética , Mutação Puntual/genética , Animais , Doenças Autoimunes/patologia , Biomarcadores/metabolismo , Modelos Animais de Doenças , Otopatias/genética , Feminino , Hiperplasia/genética , Hiperplasia/patologia , Hipofosfatemia/genética , Hipofosfatemia/patologia , Interleucina-6/metabolismo , Fígado/patologia , Masculino , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos , Mutagênese/genética , Fenótipo , Fator de Transcrição STAT3/metabolismo , Dermatopatias Genéticas/genética , Baço/patologia , Subpopulações de Linfócitos T , Trombocitopenia/genéticaRESUMO
Over the last two decades, increased availability of human pancreatic tissues has allowed for major expansions in our understanding of islet biology in health and disease. Indeed, studies of fixed and frozen pancreatic tissues, as well as efforts using viable isolated islets obtained from organ donors, have provided significant insights toward our understanding of diabetes. However, the procedures associated with islet isolation result in distressed cells that have been removed from any surrounding influence. The pancreas tissue slice technology was developed as an in situ approach to overcome certain limitations associated with studies on isolated islets or fixed tissue. In this Perspective, we discuss the value of this novel platform and review how pancreas tissue slices, within a short time, have been integrated in numerous studies of rodent and human islet research. We show that pancreas tissue slices allow for investigations in a less perturbed organ tissue environment, ranging from cellular processes, over peri-islet modulations, to tissue interactions. Finally, we discuss the considerations and limitations of this technology in its future applications. We believe the pancreas tissue slices will help bridge the gap between studies on isolated islets and cells to the systemic conditions by providing new insight into physiological and pathophysiological processes at the organ level. ARTICLE HIGHLIGHTS: Human pancreas tissue slices represent a novel platform to study human islet biology in close to physiological conditions. Complementary to established technologies, such as isolated islets, single cells, and histological sections, pancreas tissue slices help bridge our understanding of islet physiology and pathophysiology from single cell to intact organ. Diverse sources of viable human pancreas tissue, each with distinct characteristics to be considered, are available to use in tissue slices for the study of diabetes pathogenesis.
Assuntos
Diabetes Mellitus , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Humanos , Pâncreas , Doadores de TecidosRESUMO
Insulin-producing ß-cells in pancreatic islets are regulated by systemic cues and, locally, by adjacent islet hormone-producing 'non-ß-cells' (namely α-cells, δ-cells and γ-cells). Yet whether the non-ß-cells are required for accurate insulin secretion is unclear. Here, we studied mice in which adult islets are exclusively composed of ß-cells and human pseudoislets containing only primary ß-cells. Mice lacking non-ß-cells had optimal blood glucose regulation, enhanced glucose tolerance, insulin sensitivity and restricted body weight gain under a high-fat diet. The insulin secretion dynamics in islets composed of only ß-cells was comparable to that in intact islets. Similarly, human ß-cell pseudoislets retained the glucose-regulated mitochondrial respiration, insulin secretion and exendin-4 responses of entire islets. The findings indicate that non-ß-cells are dispensable for blood glucose homeostasis and ß-cell function. These results support efforts aimed at developing diabetes treatments by generating ß-like clusters devoid of non-ß-cells, such as from pluripotent stem cells differentiated in vitro or by reprograming non-ß-cells into insulin producers in situ.
RESUMO
Histopathological heterogeneity in the human pancreas is well documented; however, functional evidence at the tissue level is scarce. Herein, we investigate in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in donors without diabetes (ND; n = 15), positive for one islet autoantibody (1AAb+; n = 7), and with type 1 diabetes (T1D; <14 months duration, n = 5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features are comparable across regions in ND. In T1D, insulin secretion and beta-cell volume are significantly reduced within all regions, while glucagon and enzymes are unaltered. Beta-cell volume is lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ are consistent across the PH, PB, and PT. This study supports low inter-regional variation in pancreas slice function and, potentially, increased metabolic demand in 1AAb+.
Assuntos
Diabetes Mellitus Tipo 1 , Insulina , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Insulina/metabolismo , Feminino , Secreção de Insulina/efeitos dos fármacos , Adulto , Pessoa de Meia-Idade , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Glucagon/metabolismo , Glucose/metabolismo , Autoanticorpos/imunologia , Amilases/metabolismoRESUMO
The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.
Assuntos
Diferenciação Celular , Genes Reporter , Células Secretoras de Insulina , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Linhagem Celular , Insulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genéticaRESUMO
Histopathological heterogeneity in human pancreas has been well documented; however, functional evidence at the tissue level is scarce. Herein we investigated in situ glucose-stimulated islet and carbachol-stimulated acinar cell secretion across the pancreas head (PH), body (PB), and tail (PT) regions in no diabetes (ND, n=15), single islet autoantibody-positive (1AAb+, n=7), and type 1 diabetes donors (T1D, <14 months duration, n=5). Insulin, glucagon, pancreatic amylase, lipase, and trypsinogen secretion along with 3D tissue morphometrical features were comparable across the regions in ND. In T1D, insulin secretion and beta-cell volume were significantly reduced within all regions, while glucagon and enzymes were unaltered. Beta-cell volume was lower despite normal insulin secretion in 1AAb+, resulting in increased volume-adjusted insulin secretion versus ND. Islet and acinar cell secretion in 1AAb+ were consistent across PH, PB and PT. This study supports low inter-regional variation in pancreas slice function and potentially, increased metabolic demand in 1AAb+.
RESUMO
Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1(Aga2/+), animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6-11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.
Assuntos
Colágeno Tipo I/genética , Hemorragia/diagnóstico , Imagem Óptica/métodos , Osteogênese Imperfeita/diagnóstico , Tomografia Computadorizada por Raios X/métodos , Animais , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Fluorescência , Hemorragia/etiologia , Camundongos , Camundongos Mutantes , Osteogênese Imperfeita/complicações , Osteogênese Imperfeita/fisiopatologia , TóraxRESUMO
The mouse is a valuable model organism for studying bone biology and for unravelling pathological processes in skeletal disorders. In vivo methods like X-ray analysis, DXA measurements, pQCT and µCT are available to investigate the bone phenotype of mutant mice. However, the descriptive nature of such methods does not provide insights into the cellular and molecular bases of the observed bone alterations. Thus, first-line investigations might be complemented by cell culture-based methods to characterize the pathological processes at the cellular level independent from systemic influences. By combining well-established assays, we designed a comprehensive test system to investigate the cellular and molecular phenotype of primary calvarial osteoblasts in mutant mice compared to wild-type controls as a first-line phenotyping method. The compilation of 9 different quantifiable assays allows assessment of general properties of cell growth and investigation of bone-specific parameters at the functional, protein and RNA level in a kinetic fashion throughout a 3-week culture period, thus maximizing the chance to discover and explain new phenotypes in mutant mice. By analyzing mutant mouse lines for Col1a1 and Jag1 (Delta-Notch pathway) that both showed clear alterations in several bone-related parameters we could demonstrate the usefulness of our cell culture system to discriminate between primary (Col1a1) and secondary effects (Jag1) in osteoblasts.
Assuntos
Osso e Ossos/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Colágeno Tipo I/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Osteoblastos/patologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Cadeia alfa 1 do Colágeno Tipo I , Fêmur/diagnóstico por imagem , Fêmur/patologia , Regulação da Expressão Gênica , Proteína Jagged-1 , Camundongos , Camundongos Mutantes , Osteoblastos/metabolismo , Fenótipo , Padrões de Referência , Reprodutibilidade dos Testes , Proteínas Serrate-Jagged , Tomografia Computadorizada por Raios XAssuntos
Proteínas de Fluorescência Verde/genética , Insulina/genética , Ilhotas Pancreáticas/fisiologia , Pâncreas/citologia , Pâncreas/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Regulação da Expressão Gênica/genética , Glucose/farmacologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/embriologia , Sus scrofa , SuínosRESUMO
Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.
Assuntos
Doenças Ósseas Metabólicas/genética , Modelos Animais de Doenças , Camundongos/genética , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/enzimologia , Cálcio/sangue , Cromossomos de Mamíferos , Análise Mutacional de DNA , Etilnitrosoureia/farmacologia , Feminino , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutagênese , Mutagênicos/farmacologia , Mutação , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Fenótipo , Fosfatos/sangue , Polimorfismo de Nucleotídeo Único , Receptores de Detecção de Cálcio/genética , Estatísticas não Paramétricas , Cromossomo XRESUMO
Under the label of the German Mouse Clinic (GMC), a concept has been developed and implemented that allows the better understanding of human diseases on the pathophysiological and molecular level. This includes better understanding of the crosstalk between different organs, pleiotropy of genes, and the systemic impact of envirotypes and drugs. In the GMC, experts from various fields of mouse genetics and physiology, in close collaboration with clinicians, work side by side under one roof. The GMC is an open-access platform for the scientific community by providing phenotypic analysis in bilateral collaborations ("bottom-up projects") and as a partner and driver in international large-scale biology projects ("top-down projects"). Furthermore, technology development is a major topic in the GMC. Innovative techniques for primary and secondary screens are developed and implemented into the phenotyping pipelines (e.g., detection of volatile organic compounds, VOCs).
Assuntos
Modelos Animais , Animais , Alemanha , Camundongos , FenótipoRESUMO
Personalized in vitro models for dysplasia and carcinogenesis in the pancreas have been constrained by insufficient differentiation of human pluripotent stem cells (hPSCs) into the exocrine pancreatic lineage. Here, we differentiate hPSCs into pancreatic duct-like organoids (PDLOs) with morphological, transcriptional, proteomic, and functional characteristics of human pancreatic ducts, further maturing upon transplantation into mice. PDLOs are generated from hPSCs inducibly expressing oncogenic GNAS, KRAS, or KRAS with genetic covariance of lost CDKN2A and from induced hPSCs derived from a McCune-Albright patient. Each oncogene causes a specific growth, structural, and molecular phenotype in vitro. While transplanted PDLOs with oncogenic KRAS alone form heterogenous dysplastic lesions or cancer, KRAS with CDKN2A loss develop dedifferentiated pancreatic ductal adenocarcinomas. In contrast, transplanted PDLOs with mutant GNAS lead to intraductal papillary mucinous neoplasia-like structures. Conclusively, PDLOs enable in vitro and in vivo studies of pancreatic plasticity, dysplasia, and cancer formation from a genetically defined background.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Células-Tronco Pluripotentes , Animais , Humanos , Camundongos , Mutação , Organoides , Ductos Pancreáticos , Neoplasias Pancreáticas/genética , ProteômicaRESUMO
Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.
Assuntos
Câmara Anterior/anatomia & histologia , Transplante das Ilhotas Pancreáticas/diagnóstico por imagem , Transplante das Ilhotas Pancreáticas/métodos , Microscopia Confocal/métodos , Animais , Câmara Anterior/transplante , Câmara Anterior/ultraestrutura , Modelos Animais de Doenças , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Transplante das Ilhotas Pancreáticas/instrumentação , Estudos Longitudinais , Camundongos , Camundongos Mutantes , Microscopia Confocal/instrumentação , Transplante HeterotópicoRESUMO
Studies on islet of Langerhans physiology are crucial to understand the role of the endocrine pancreas in diabetes pathogenesis and the development of new therapeutic approaches. However, so far most research addressing islet of Langerhans biology relies on islets obtained via enzymatic isolation from the pancreas, which is known to cause mechanical and chemical stress, thus having a major impact on islet cell physiology. To circumvent the limitations of islet isolation, we have pioneered a platform for the study of islet physiology using the pancreas tissue slice technique. This approach allows to explore the detailed three-dimensional morphology of intact pancreatic tissue at a cellular level and to investigate islet cell function under near-physiological conditions. The described procedure is less damaging and faster than alternative approaches and particularly advantageous for studying infiltrated and structurally damaged islets. Furthermore, pancreas tissue slices have proven valuable for acute studies of endocrine as well as exocrine cell physiology in their conserved natural environment. We here provide a detailed protocol for the preparation of mouse pancreas tissue slices, the assessment of slice viability, and the study of pancreas cell physiology by hormone secretion and immunofluorescence staining.
Assuntos
Técnicas de Preparação Histocitológica/métodos , Ilhotas Pancreáticas/fisiologia , Pâncreas/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Sobrevivência Celular/fisiologia , Imunofluorescência/métodos , Secreção de Insulina/fisiologia , Camundongos , Técnicas Analíticas Microfluídicas/métodos , Coleta de Tecidos e ÓrgãosRESUMO
Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify ß-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and ß-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in ß-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis.
Assuntos
Glicemia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Monitorização Fisiológica , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos SCID , Peptídeos/metabolismoRESUMO
Type 2 diabetes is characterized by peripheral insulin resistance and insufficient insulin release from pancreatic islet ß cells. However, the role and sequence of ß cell dysfunction and mass loss for reduced insulin levels in type 2 diabetes pathogenesis are unclear. Here, we exploit freshly explanted pancreas specimens from metabolically phenotyped surgical patients using an in situ tissue slice technology. This approach allows assessment of ß cell volume and function within pancreas samples of metabolically stratified individuals. We show that, in tissue of pre-diabetic, impaired glucose-tolerant subjects, ß cell volume is unchanged, but function significantly deteriorates, exhibiting increased basal release and loss of first-phase insulin secretion. In individuals with type 2 diabetes, function within the sustained ß cell volume further declines. These results indicate that dysfunction of persisting ß cells is a key factor in the early development and progression of type 2 diabetes, representing a major target for diabetes prevention and therapy.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Insulina/patologia , Idoso , Glicemia/metabolismo , Feminino , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Pâncreas/metabolismoRESUMO
In type 1 diabetes (T1D), autoimmune destruction of pancreatic ß cells leads to insulin deficiency and loss of glycemic control. However, knowledge about human pancreas pathophysiology in T1D remains incomplete. To address this limitation, we established a pancreas tissue slice platform of donor organs with and without diabetes, facilitating the first live cell studies of human pancreas in T1D pathogenesis to our knowledge. We show that pancreas tissue slices from organ donors allow thorough assessment of processes critical for disease development, including insulin secretion, ß cell physiology, endocrine cell morphology, and immune infiltration within the same donor organ. Using this approach, we compared detailed pathophysiological profiles for 4 pancreata from donors with T1D with 19 nondiabetic control donors. We demonstrate that ß cell loss, ß cell dysfunction, alterations of ß cell physiology, and islet infiltration contributed differently to individual cases of T1D, allowing insight into pathophysiology and heterogeneity of T1D pathogenesis. Thus, our study demonstrates that organ donor pancreas tissue slices represent a promising and potentially novel approach in the search for successful prevention and reversal strategies of T1D.