Hyperglycaemia and its implication on the Pancreatic islet microvasculature in diabetic rat models

Ngounou, Eleonore (2020-12)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

SUMMARY BACKGROUND: Despite the considerable progress made in the treatment of diabetes mellitus, vascular damage remains the leading cause of patient death. The mechanisms underlying vascular abnormalities in obesity and diabetes mellitus remain to be elucidated and may be the main cause of β-cell death. In addition, the detailed description of islet microvasculature in the pancreas is lacking in the literature; therefore, a better understanding of the characteristics of the blood vessel and the factors that maintain β-cell function is needed in clinical practice. OBJECTIVE: To describe the spatial distribution and histomorphology of islet microvasculature under the effect of hyperglycaemia in two experimental diabetic models. METHODS: Eight week old male Wistar rats (n=50) were divided into two groups that received either a standard diet (RAC) (n=20) or a high-fat diet (HFD) (n=30) for two weeks. By the end of the two weeks, altered glucose uptake was confirmed in the HFD group by an oral glucose tolerance test (OGTT). A subgroup (RAC / STZ) of the RAC group (n=10) and another (HFD / STZ) of the HFD group (n=10) then received 50 and 35mg/kg of body weight (BW) of streptozotocin (STZ) to induce type I diabetes mellitus and type II diabetes mellitus, respectively. They were kept diabetic for an additional eight weeks. The body weight and blood glucose (BGL) of the animals were recorded throughout the experimental period (88 days). Blood was collected for flow cytometry and Luminex assay before half the number of animals were sacrificed for pancreatic tissue collection for histological procedure. The remaining half was used to replicate (cast) the pancreatic vasculature by perfusion with polyurethane-based casting resin (PU4ii). Haematoxylin and Eosin (H&E) stained sections were used to assess the general morphology of pancreatic tissue. Methenamine silver and immunostaining using CD34 antibody, delineated the basement membrane and endothelial cells, respectively, of islet microvasculature. A digital camera and a nano-computed tomography (nano-CT) scanner made it possible to generate digital and 3D images. Quantitative evaluation of topographic morphometric parameters of the pancreatic vascular network in the duodenal and splenic regions of the pancreas in each experimental condition was performed using the imageJ and Volume Graphics VGStudioMax 3.0®. Reconstruction of the pancreatic vascular network was attempted using the vascular tree scale laws. RESULTS: A significant increase in the mean body weight was accompanied by a slight increase in mean BGL within 2 weeks in HFD. Streptozotocin caused the development of two diabetic models with all clinical symptoms (polyuria, polyphagia, high BGL (> 28mmol/L) and a significant decrease in body mass in both diabetic groups (26.68% and 15.54% in RAC / STZ and HFD / STZ respectively). The results of the flow cytometry and the Luminex assay validated the presence of islet vascular lesions in animals, which also justified the significant necrosis of endothelial cells, a decrease (p<0.05) in the mean percentage of the stained area of CD34 pixels in islets, and thickening of the basement membrane. The scaling law was used to obtain the relationships between 1) the length and volume of the pancreatic vascular tree up to capillary level (R2=0.693±0.053), 2) the diameter of the lumen and the blood flow in each pancreatic vascular branch (R2=0.988±0.055), and 3) the diameter and length of the branches of the vessels (R2=0.838±0.0123). CONCLUSION: This investigation has established detailed morphological features of the vasculature of the pancreas in the duodenal and splenic regions in normal and diabetic rat models. There were large differences in the structure of the pancreatic vasculature between the two regions appearing to be dictated by metabolic demand. However, there are still challenges in 3D visualisation of the capillary networks of the pancreatic vascular tree, which was the main limitation of this study.

AGTERGROND: Ten spyte van aansienlike vooruitgang in behandeling van diabetes mellitus, bly vaskulêre skade die hoof oorsaak van pasiënt sterftes. Die meganismes onderliggend aan vaskulêre abnormaliteite in vetsugtigheid en diabetes mellitus moet uitgelig word, want dit mag die hoof oorsaak van afsterwing van β-selle wees. Tesame hiermee, is daar ook ‘n gebrek aan ‘n breedvoerige beskrywing wat handel oor die mikrovaskulatuur in die pankreas in die literatuur; met ander woorde, verbetering in kennis van die eienskappe van die bloedvate en die faktore wat β-sel funksie handhaaf, word in die kliniese praktyk benodig. DOELWIT: Om die effek van hiperglisemie op die ruimtelike verspreiding en histomorfologie van eiland mikrovaskulatuur in twee eksperimentele diabetiese modelle te beskryf. METODES: Agt weke oue manlike Wistar rotte (n=50) is vir twee weke volgens die rotte se diëte, onderskeidelik ‘n standaard (RAC) dieët (n=20), en ‘n hoë vet (HFD) dieët (n=30). ‘n Verminderde glukose opname is na twee weke in die HFD groep bevestig deur gebruik te maak van ‘n orale glukose toleransie toets (OGTT). ‘n Subgroep van die standaard dieët (RAC/STZ) (n =10) en die hoë vet dieët (HFD/STZ) (n=10), het daarna 50 en 35mg/kg liggaamsgewig (BW) Streptozotocin (STZ) ontvang om tipe I diabetes mellitus en tipe II diabetes mellitus, onderskeidelik, te induseer. Die rotte is vir ‘n verdere agt weke in ‘n diabetiese toestand gehou. Daar is deur die eksperimentele periode (88 dae), rekord gehou van die liggaamsmassa en bloedglukosevlakke (BGL) van die diere. Bloed is vir sitometrie en Luminex bepaling getrek voor die helfte van die aantal diere geoffer is vir verkryging van pankreasweefsel vir histologiese prosedures. Deur gebruik van perfusie met ‘n poliuretraan gebaseerde giet hars (PU4ii) is weefselafgietsels van die bloedvate van die pankreas in die oorblywende helfte gemaak. Hematoksillien en Eosien (H&E) kleuring is gebruik om die algemene morfologie van die pankreasweefsel te bepaal. Met behulp van Methenamine silver, en immunositochemiese kleuring met behulp van CD34 teenliggame, is die basaalmembraan en endoteelselle van die eiland mikrovaskulatuur, onderskeidelik, geïllustreer. ‘n Digitale kamera en nano-CT skandeerder het dit moontlik gemaak om digitale en 3D beelde te skep. Met behulp van ImageJ en Volume Graphics VGStudioMax 3.0® is kwantitatiewe evaluering van topografiese morfometriese kenmerke van die vaskulêre netwerk in die duodenale - en spleniese areas van die pankreas in elke eksperimentele toestand gedoen. Vaskulêre boom skaaltoepassings is gebruik vir rekonstruksie van die vaskulêre netwerk van die pankreas. RESULTATE: In diere met ‘n hoë vet dieët (HFD) is binne 2 weke ‘n betekenisvolle toename in gemiddelde liggaamsmassa, asook ‘n geringe toename in gemiddelde bloedglukosevlakke (BGL), waargeneem. Na toediening van Streptozotocin is twee diabetiese modelle wat alle kliniese simptome insluit (poliurie, polifagie, hoë bloedglukosevlakke (>28mmol/L) ontwikkel, asook ‘n betekenisvolle afname in liggaamsmassa in beide diabetiese groepe (26.68% en 15.54% in RAC / STZ en HFD / STZ, onderskeidelik). Resultate na vloeisitometrie en Luminex bepaling het die teenwoordigheid van vaskulêre letsels in die eilande van die diere, asook betekenisvolle nekrose in endoteelselle, ‘n afname (p<0.05) in die gemiddelde persentasie in die area met CD34 kleuring in die eiland, en verdikking van die basaalmembraan, bevestig. Skaaltoepassings het die verhouding tussen 1) die lengte en vaskulêre volume van die vaskulêre boom van die pankreas tot op kapillêre vlak (R2=0.693±0.053), 2) die deursnit van die lumen en bloedvloei in elke vaskulêre tak in die pankreas (R2=0.988±0.055), en 3) die deursnit en lengte van die takke van die bloedvate (R2=0.838±0.0123), bevestig. KONKLUSIE: Hierdie navorsing het gedetailleerde morfologiese eienskappe van die vaskulatuur van die pankreas in beide die duodenale en spleniese gedeeltes in normale en diabetiese rotmodelle bevestig. Daar bestaan groot verskille in die struktuur van die bloedvate tussen die twee gebiede in die pankreas, wat klaarblyklik metaboliese aanvraag weerspieël. Daar is egter nog uitdagings in 3D beelding van die kapillêre netwerke in die vaskulêre boom van die pankreas. Laasgenoemde was dan ook die grootste beperking van die studie.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/109311
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