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Quantification and 3-D imaging of the insulitis-induced destruction of β-cells in murine type 1 diabetes
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). (Ahlgren Ulf)
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). (Ahlgren Ulf)
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics. (Holmberg Dan)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2010 (English)In: Diabetes, ISSN 0012-1797, E-ISSN 1939-327X, Vol. 59, no 7, 1756-1764 p.Article in journal (Refereed) Published
Abstract [en]

Objective: The aim of this study was to refine the information regarding the quantitative and spatial dynamics of infiltrating lymphocytes and remaining beta-cell volume during the progression of type 1 diabetes in the NOD mouse model of the disease.

Research design and methods: Using an ex vivo technique, optical projection tomography (OPT), we quantified and assessed the 3D spatial development and progression of insulitis and beta-cell destruction in pancreas from diabetes prone NOD and non-diabetes prone congenic NOD.H-2b mice between 3 and 16 weeks of age.

Results: Together with results showing the spatial dynamics of the insulitis process we provide data of beta-cell volume distributions down to the level of the individual islets and throughout the pancreas during the development and progression of type 1 diabetes. Our data provide evidence for a compensatory growth potential of the larger insulin(+) islets during the later stages of the disease around the time point for development of clinical diabetes. This is in contrast to smaller islets, which appear less resistant to the autoimmune attack. We also provide new information on the spatial dynamics of the insulitis process itself, including its apparently random distribution at onset, the local variations during its further development, and the formation of structures resembling tertiary lymphoid organs at later phases of insulitis progression.

Conclusions: Our data provides a powerful tool for phenotypic analysis of genetic and environmental effects on type 1 diabetes etiology as well as for evaluating the potential effect of therapeutic regimes.

Place, publisher, year, edition, pages
2010. Vol. 59, no 7, 1756-1764 p.
Keyword [en]
pancreas, mouse, mice
National Category
Endocrinology and Diabetes
Identifiers
URN: urn:nbn:se:umu:diva-33656DOI: 10.2337/db09-1400ISI: 000279615100025PubMedID: 20393145OAI: oai:DiVA.org:umu-33656DiVA: diva2:316462
Available from: 2010-04-30 Created: 2010-04-30 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Optical projection tomography based 3D-spatial and quantitative assessments of the diabetic pancreas
Open this publication in new window or tab >>Optical projection tomography based 3D-spatial and quantitative assessments of the diabetic pancreas
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The gastrointestinal tract comprises a number of digestive organs including the stomach and pancreas. The stomach is involved in the digestion and short term storage of food while the pancreas is a mixed endocrine and exocrine gland which provides the body with hormones and enzymes essential for nutritional utilisation. The pancreas consists of three different cell lineages, acinar, ductal and endocrine cells. The endocrine cells, organised in the islets of Langerhans, are scattered throughout the exocrine parenchyma and regulate blood glucose levels by production of hormones such as glucagon and insulin.

The Nkx family of homeodomain proteins controls numerous processes during development. Previous studies have identified two members belonging to the Nkx6 subfamily of Nkx proteins, Nkx6.1 and Nkx6.2. We have described the cloning and embryonic expression pattern of Nkx6.3. All three members of the Nkx6 gene family were shown to be expressed in partially overlapping domains during the development of the gastrointestinal tract and the central nervous system. Nkx6.2 was also identified as a transient marker for pancreatic exocrine cells.

Analysing gene expression patterns and morphological features in tissues and organs is often performed by stereologic sampling which is a labour-intensive two dimensional approach that rely on certain assumptions when calculating e.g. β-cell mass and islet number in the pancreas. By combined improvements in immunohistochemical protocols, computational processing and tomographic scanning, we have developed a methodology based on optical projection tomography (OPT) allowing for 3D visualisation and quantification of specifically labelled objects within intact adult mouse organs. In the pancreas, this technique allows for spatial and quantitative measurements of total islet number and β-cell mass. We have further developed a protocol allowing for high resolution regional analyses based on global OPT assessments of the pancreatic constitution. This methodology is likely to facilitate detailed cellular and molecular analysis of user defined regions of interest in the pancreas, at the same time providing information on the overall disease state of the gland.

Type 1 diabetes mellitus (T1D) can occur at any age and is characterized by the marked inability of the pancreas to secrete insulin due to an autoimmune destruction of the insulin producing β-cells. Information on the key cellular and molecular events underlying the recruitment of lymphocytes, their infiltration of the islets of Langerhans and consequent β-cell destruction is essential for understanding the pathogenesis of T1D. Using the developed methodology we have recorded the spatial and quantitative distribution of islet β-cells and infiltrating lymphocytes in the non obese diabetic (NOD) mouse model for T1D. This study shows that the smaller islets, which are predominantly organised in the periphery of the organ, are the first to disappear during the progression of T1D. The larger islets appear more resistant and our data suggest that a compensatory proliferative process is going on side by side with the autoimmune-induced β-cell destruction. Further, the formation of structures resembling tertiary lymphoid organs (TLOs) in areas apparently unaffected by insulitis suggests that local factors may provide cues for the homing of these lymphocytes back to the pancreas.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär medicin (UCMM), 2008. 53 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1232
Keyword
Nkx, development, optical projection tomography, 3D visualisation, pancreas, beta-cell mass, insulitis, diabetes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1939 (URN)978-91-7264-700-8 (ISBN)
Public defence
2008-12-13, Betula, 6M, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-11-25 Created: 2008-11-25 Last updated: 2011-04-05Bibliographically approved
2. Imaging the pancreas: new aspects on lobular development and adult constitution
Open this publication in new window or tab >>Imaging the pancreas: new aspects on lobular development and adult constitution
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mouse pancreas is a mixed exocrine and endocrine glandconsisting of three lobular compartments: the splenic, duodenal and gastric lobes. During embryogenesis, the pancreas forms from two progenitor populations located on the dorsal and ventral side of the primitive gut tube. These anlagen are brought in close proximity as the gut elongates and rotates, and fuse to form a single organ. The splenic and duodenal lobes develop from the dorsal and ventral anlagen, respectively.

In the adult pancreas, exocrine tissue secretes digestive enzymes intothe gut lumen to support nutrient uptake. The endocrine Islets of Langerhans are scattered throughout the exocrine tissue and aid in regulation of energy homeostasis through the secretion of hormones. One of the key players in energy homeostasis is the pancreatic ß-cell, which is the most abundant cell type of the islets. The β-cells regulates blood glucose levels through the action of insulin. Conditions where this regulation does not function properly are gathered under the common name of Diabetes mellitus.

Type 1 diabetes (T1D) is characterized by insulin deficiency due to autoimmune destruction of the ß-cells. Using recently developed protocols for optical projection tomography (OPT) whole-organ imaging, we have revealed new spatial and quantitative aspects on ß-cell mass dynamics and immune infiltration during the course of T1D development in the non-obese diabetic (NOD) mouse model. We show that although immune infiltration appears to occur asynchronously throughout the organ, smaller islets, mainly located in the periphery of the organ, preferentially loose their ß-cells during early stages of disease progression. Larger islets appear more resistant to the autoimmune attack and our data indicate the existence of a compensatory proliferative capacity within these islets. We also report the appearance of structures resembling tertiary lymphoid organs (TLOs) in association with the remaining islets during later phases of T1D progression.

OPT has already proven to be a useful tool for assessments of ß-cellmass in the adult mouse pancreas. However, as with other techniques, previous protocols have relied on a tedious degree of manual postivacquisition editing. To further refine OPT-based assessment of pancreatic ß-cell mass distribution in the murine pancreas, we implemented a computational statistical approach, Contrast-Limited Adaptive Histogram Normalisation (CLAHE), to the OPT projection data of pancreata from C57Bl/6 mice. This methodology provided increased islet detection sensitivity, improved islet morphology and diminished subjectivity in thresholding for reconstruction and quantification. Using this approach, we could report a substantially higher number of islets than previously described for this strain and provide evidence of significant differences in islet mass distribution between the pancreatic lobes. The gastric lobe stood out in particular and contained a 75% higher islet density as compared to the splenic lobe.

Although the development of the early pancreatic buds has been relatively well studied, later morphogenetic events are less clear and information regarding the formation of the gastric lobe has largely been missing. Using OPT we have generated a quantitative three-dimensional road map of pancreatic morphogenesis in the mouse. We show that the gastric lobe forms as a perpendicular outgrowth fromthe stem of the dorsal pancreas at around embryonic day (e) 13.5, which grows into a mesenchymal domain overlaying the pyloric sphincter and proximal part of the glandular stomach. By analyzing mutant mice with aberrant spleen development, we further demonstrate that proper formation of the gastric lobe is dependent on the initial formation of the closely positioned spleen, indicating a close interplay between pancreatic and splenic mesenchyme during development. Additionally, we show that the expression profile of markers for pancreatic multipotent progenitors within the pancreas is heterogenous with regards to lobular origin. Altogether, our studies regarding the morphogenesis and adult constitution of the mouse pancreas recognize lobular heterogeneities that add important information for future interpretations of this organ.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2011. 50 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1471
Keyword
Type 1 diabetes, ß-cell mass, tertiary lymphoid organs, biomedical imaging, optical projection tomography, morphogenesis, pancreas development, gastric lobe, spleen development.
National Category
Other Basic Medicine Medical Genetics
Research subject
Medical Genetics; Developmental Neurosciences; Molecular Medicine
Identifiers
urn:nbn:se:umu:diva-50601 (URN)978-91-7459-341-9 (ISBN)
Public defence
2012-01-20, Betula, Analysvägen 1, By 6M, Vån 1, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-12-22 Created: 2011-12-15 Last updated: 2011-12-22Bibliographically approved

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Alanentalo, TomasHörnblad, AndreasMayans, SofiaNilsson, Anna KarinLarefalk, ÅsaAhlgren, UlfHolmberg, Dan
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