The Role of Exocrine Pancreatic Inflammation in the ...
1
The Role of Exocrine Pancreatic Inflammation in the Pathogenesis of Canine Diabetes Mellitus INTRODUCTION METHODS RESULTS CONCLUSIONS ACKNOWLEDGEMENTS • Students Training in Advanced Research (STAR) Program: NIH T35 OD010956-19 grant • Center for Companion Animal Health (CCAH), UC Davis • *Shared senior authorship Diabetes Mellitus (DM) is a chronic and debilitating metabolic disease that affects hundreds of thousands of dogs worldwide. Canine DM is characterized by pancreatic β-cell loss, leading to insulin deficiency and persistent hyperglycemia. DM and exocrine pancreatic inflammation (pancreatitis) are closely associated, and spillover inflammation from pancreatitis to neighboring endocrine tissue and collateral pancreatic endocrine tissue injury has been proposed as a potential etiology for canine DM. Direct collateral damage from pancreatitis in dogs results in proportional destruction of pancreatic α and β cells. • Marked islet hypoplasia with beta cell preferential loss noted in DM only samples so far • No CD45 detected in analyzed islet images of healthy and DM only samples • Confocal immunofluorescence protocol optimized for efficient, high quality images of whole tissue sections and individual islets • Preliminary result acquisition and analysis in progress While image analysis is ongoing, early results show a difference in the number, size, and composition of islets between DM and healthy controls. Healthy pancreas samples have hundreds of islets with large islet size (>20 cells). By comparison, DM only samples have few islets but plenty of individual endocrine cells present. DM only samples have very few insulin+ cells and varying levels of glucagon+ cells. No CD45 has been noted in or around selected islets thus far. Staining • Antigen Retrieval Image Acquisition • Confocal Immunofluorescence Image Analysis • ImageJ Diabetes Mellitus Only n=10 Pancreatitis Only n=10 Concurrent Diabetes and Pancreatitis n=10 Healthy Pancreas n=5 Tissue sections from dogs were selected and categorized into one of four groups based on clinical and pathological diagnosis. Slides were stained for insulin, glucagon, CD45 (panleukocyte marker), and DAPI (nuclear counterstain). Following staining, whole tissue sections were imaged at 20x using an image capture and stitching technique. Whole tissue sections comprised of hundreds to thousands of stitched tile images. 15-20 high resolution stacked images were then captured of individual islets at 63x. Images were analyzed to determine islet density per tissue section area, α:β cell ratio per islet, and number of leukocytes within and surrounding individual islets. Primary Antibodies: • Polyclonal Guinea Pig anti-insulin (DAKO) • Rabbit anti-glucagon (Cell Signaling Technology Glucagon Antibody #2760) • Mouse anti-CD45 (Peter Moore Lab, UC Davis) Secondary Antibodies: • Alexa Fluor 488 AffiniPure Donkey Anti-Guinea Pig IgG • Alexa Fluor 647 AffiniPure Donkey Anti-Rabbit IgG • Cy3 AffiniPure Donkey Anti-Mouse IgG HYPOTHESIS Figure 1. Islet counting was performed by magnifying 20x stitched tiles of whole pancreas sections and marking individual islets using ImageJ software. Rebecca Horn, Dustin Leale, Chen Gilor * , and Amir Kol * School of Veterinary Medicine, University of California, Davis Figure 2. Pancreatic islet immunofluorescence at 63x in healthy (A,B) and diabetic (C,D) dogs. DM dogs have fewer and smaller islets, as well as a higher α:β cell ratio per islet. DAPI GLU INS CD45 A C B D DAPI GLU INS CD45
Transcript of The Role of Exocrine Pancreatic Inflammation in the ...
The Role of Exocrine Pancreatic Inflammation in the Pathogenesis of Canine Diabetes Mellitus
INTRODUCTION
METHODS
RESULTS
CONCLUSIONS
ACKNOWLEDGEMENTS
• Students Training in Advanced Research (STAR) Program: NIH T35 OD010956-19 grant
• Center for Companion Animal Health (CCAH), UC Davis• *Shared senior authorship
Diabetes Mellitus (DM) is a chronic and debilitating metabolicdisease that affects hundreds of thousands of dogsworldwide. Canine DM is characterized by pancreatic β-cellloss, leading to insulin deficiency and persistenthyperglycemia. DM and exocrine pancreatic inflammation(pancreatitis) are closely associated, and spilloverinflammation from pancreatitis to neighboring endocrinetissue and collateral pancreatic endocrine tissue injury hasbeen proposed as a potential etiology for canine DM.
Direct collateral damage from pancreatitis in dogs results inproportional destruction of pancreatic α and β cells.
• Marked islet hypoplasia with beta cell preferential lossnoted in DM only samples so far
• No CD45 detected in analyzed islet images of healthy andDM only samples
• Confocal immunofluorescence protocol optimized forefficient, high quality images of whole tissue sections andindividual islets
• Preliminary result acquisition and analysis in progressWhile image analysis is ongoing, early results show a difference inthe number, size, and composition of islets between DM andhealthy controls. Healthy pancreas samples have hundreds of isletswith large islet size (>20 cells). By comparison, DM only sampleshave few islets but plenty of individual endocrine cells present. DMonly samples have very few insulin+ cells and varying levels ofglucagon+ cells. No CD45 has been noted in or around selectedislets thus far.
Staining
• Antigen Retrieval
Image Acquisition
• Confocal Immunofluorescence
Image Analysis
• ImageJ
Diabetes Mellitus Only
n=10
Pancreatitis Only
n=10
Concurrent Diabetes and Pancreatitis
n=10
Healthy Pancreas
n=5
Tissue sections fromdogs were selectedand categorized intoone of four groupsbased on clinical andpathological diagnosis.Slides were stained forinsulin, glucagon, CD45(panleukocyte marker),and DAPI (nuclearcounterstain).
Following staining, whole tissue sections were imaged at 20xusing an image capture and stitching technique. Whole tissuesections comprised of hundreds to thousands of stitched tileimages. 15-20 high resolution stacked images were then capturedof individual islets at 63x. Images were analyzed to determine isletdensity per tissue section area, α:β cell ratio per islet, and numberof leukocytes within and surrounding individual islets.
Primary Antibodies:• Polyclonal Guinea Pig anti-insulin (DAKO)• Rabbit anti-glucagon (Cell Signaling Technology
Glucagon Antibody #2760)• Mouse anti-CD45 (Peter Moore Lab, UC Davis)Secondary Antibodies:• Alexa Fluor 488 AffiniPure Donkey Anti-Guinea Pig IgG• Alexa Fluor 647 AffiniPure Donkey Anti-Rabbit IgG• Cy3 AffiniPure Donkey Anti-Mouse IgG
HYPOTHESIS
Figure 1. Islet counting was performedby magnifying 20x stitched tiles ofwhole pancreas sections and markingindividual islets using ImageJ software.
Rebecca Horn, Dustin Leale, Chen Gilor*, and Amir Kol*
School of Veterinary Medicine, University of California, Davis
Figure 2. Pancreatic islet immunofluorescence at 63x in healthy (A,B) and diabetic(C,D) dogs. DM dogs have fewer and smaller islets, as well as a higher α:β cell ratioper islet.
DAPI GLU INS CD45
A C
B D
DAPI GLU INS CD45
