Integrated Brain Atlas for Unbiased Mapping of Nervous System Effects Following Liraglutide Treatment

Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-Automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected

Optical projection tomography based 3D-spatial and quantitative assessments of the diabetic pancreas

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

Optical projection tomography based 3D-spatial and quantitative assessments of the diabetic pancreas

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

Mesoscopic Optical Imaging of the Pancreas : Revisiting Pancreatic Anatomy and Pathophysiology

The exocrine-endocrine multipart organization of the pancreas makes it an exceedingly challenging organ to analyze, quantitatively and spatially. Both in rodents and humans, estimates of the pancreatic cellular composition, including beta-cell mass, has been largely relying on the extrapolation of 2D stereological data originating from limited sample volumes. Alternatively, they have been obtained by low resolution non-invasive imaging techniques providing little detail regarding the anatomical organization of the pancreas and its cellular and/or molecular make up. In this mini-review, the state of the art and the future potential of currently existing and emerging high-resolution optical imaging techniques working in the mm-cm range with μm resolution, here referred to as mesoscopic imaging approaches, will be discussed regarding their contribution toward a better understanding of pancreatic anatomy both in normal conditions and in the diabetic setting. In particular, optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) imaging of the pancreas and their associated tissue processing and computational analysis protocols will be discussed in the light of their current capabilities and future potential to obtain more detailed 3D-spatial, quantitative, and molecular information of the pancreas

Illuminating the complete ß-cell mass of the human pancreas - signifying a new view on the islets of Langerhans

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research

Cloning and analysis of Nkx6.3 during CNS and gastrointestinal development

International audienceMembers of the Nkx family of homeodomain proteins are involved in a variety of developmental processes such as cell fate determination in the CNS and in the pancreas. Here we describe the cloning and developmental expression pattern of Nkx6.3, a new member of the Nkx6 subfamily of homeodomain proteins. Nkx6.3 is expressed in the developing CNS and gastro-intestinal tract. In contrast to Nkx6.1 and Nkx6.2 that are broadly expressed in ventral positions of the developing CNS, Nkx6.3 shows a remarkably selective expression in a subpopulation of differentiating V2 neurons at caudal hindbrain levels. The expression of Nkx6.3 at this level depends on the activity of other Nkx6 proteins. In the gut, Nkx6.3 is expressed in duodenal and glandular stomach endoderm and at the end of gestation Nkx6.3 became restricted to the base of the gastric units in the glandular stomach. The expression of Nkx6.3 overlapped with the expression of Nkx6.2 both in the CNS and in the gut. Transient Nkx6.2 expression was also detected in the developing pancreas. However, analysis of Nkx6.2(-/-) mice did not display any obvious aberrations of pancreatic or stomach development

Illuminating the complete ß-cell mass of the human pancreas - signifying a new view on the islets of Langerhans

Pancreatic islets of Langerhans play a pivotal role in regulating blood glucose homeostasis, but critical information regarding their mass, distribution and composition is lacking within a whole organ context. Here, we apply a 3D imaging pipeline to generate a complete account of the insulin-producing islets throughout the human pancreas at a microscopic resolution and within a maintained spatial 3D context. These data show that human islets are far more heterogenous than previously accounted for with regards to their size distribution and cellular make up. By deep tissue 3D imaging, this in-depth study demonstrates that 50% of the human insulin-expressing islets are virtually devoid of glucagon-producing α-cells, an observation with significant implications for both experimental and clinical research

3D imaging of human organs with micrometer resolution-applied to the endocrine pancreas

The possibility to quantitatively study specific molecular/cellular features of complete human organs with preserved spatial 3D context would have widespread implications for pre-clinical and clinical medicine. Whereas optical 3D imaging approaches have experienced a formidable revolution, they have remained limited due to current incapacities in obtaining specific labelling within large tissue volumes. We present a simple approach enabling reconstruction of antibody labeled cells within entire human organs with preserved organ context. We demonstrate the utility of the approach by providing volumetric data and 3D distribution of hundreds of thousands of islets of Langerhans within the human pancreas. By assessments of pancreata from non-diabetic and type 2 diabetic individuals, we display previously unrecognized features of the human islet mass distribution and pathology. As such, this method may contribute not only in unraveling new information of the pancreatic anatomy/pathophysiology, but it may be translated to essentially any antibody marker or organ system. Hahn et al. present a method to visualize the endocrine human pancreas in 3D and calculate volumetric data. Using immunolabeling to visualize targets of interest and in reconstructing large tissue parts from imaged cm3-sized tissue blocks, they use their method to reveal previously unknown morphological differences in the endocrine pancreas affected with type 2 diabetes

Quantification and three-dimensional imaging of the insulitis-induced destruction of ß-cells in murine type 1diabetes

OBJECTIVE: The aim of this study was to refine the information regarding the quantitative and spatial dynamics of infiltrating lymphocytes and remaining β-cell volume during the progression of type 1 diabetes in the nonobese diabetic (NOD) mouse model of the disease. RESEARCH DESIGN AND METHODS: Using an ex vivo technique, optical projection tomography (OPT), we quantified and assessed the three-dimensional spatial development and progression of insulitis and β-cell destruction in pancreata 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 β-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 provide 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