Fig 1.
Pancreas inflammation in diabetes-induced rats occurs prior to insulitis while islet morphology is normal.
(A) Pancreas tissue was collected from pIC+KRV treated and control rats at the time points indicated and processed for histology and immunostaining. (B) Representative images of H&E stained pancreas sections from pIC+KRV treated and control rats. n = 6 rats/time point; scale bars, 100 um. Arrows indicate vessel/duct dilation. Representative immunofluorescent images of islets (C) and exocrine tissue (D) from pancreas sections of pIC+KRV treated and control rats; CD8 (red), insulin (green), DNA (blue). n = 4 rats/time point; scale bars, 50 um. Arrows indicate CD8+ cells.
Fig 2.
CD8+ cells infiltrate the pancreas at a very early time point after diabetes induction.
Pancreas infiltrating immune cells were analyzed at the indicated times from control rats (red circles) and pIC+KRV treated rats (blue squares) by flow cytometry. (A) Representative flow gating for pancreatic supernatant on Day 1. Single, live cells were gated based on expression of CD45; CD45+ cells were further gated on CD3 expression. CD3+ cells were divided into CD4+ and CD8+ cells and expression of CD161 was further analyzed on CD3+CD8+ cells. Additionally, CD45+ cells that were CD3-CD161high were further analyzed for expression of CD8. Shown are percentages of CD8+CD3+ cells of the CD45+ population (B), CD3+CD8+CD161+ NKT cells (C), and CD3-CD161highCD8+ NK cells (D): n = 6, each dot represents data from a single rat. Comparison is between control and pIC+KRV treated rats at each time point; *p<0.05, **p<0.01 and ***p<0.001.
Fig 3.
Caspase 3 is activated much earlier in whole pancreas than islets in diabetes-induced rats.
Protein lysates from whole pancreata or isolated islets from pIC+KRV treated and control rats were collected at the time points indicated. Representative Caspase 3 immunoblots of isolated pancreatic islets (A) and whole pancreas (C) from pIC+KRV treated and control rats; actin was used as a loading control. Densitometric analyses of the immunoblots from isolated pancreatic islets (B) and whole pancreata (D), normalized to actin. n = 3, error bars show SE. ***p<0.001 for pIC+KRV treated versus control rats.
Fig 4.
Ultrasound imaging technology can identify various blood vessels and organs, including the pancreas.
(A) Schematic drawing of major blood vessels that were used as landmarks to locate the pancreas with ultrasound imaging technology. (B-D) Visualization by ultrasound imaging technology of the pancreas, spleen and left kidney of a representative control rat using the splenic and left renal arteries as location references. Representative image using 2D imaging mode (B-C) and using 3D imaging mode (D-E).
Fig 5.
Perfusion of pancreatic micro-vasculature is highly elevated at the earliest stage following diabetes induction.
MicroMarker Contrast Imaging Mode combined with Non Linear Contrast were used to generate the ultrasound images shown. Representative ultrasound images from a treated rat are shown (A) prior to micro-bubble injection and (B) at peak of perfusion following injection. (C) Raw perfusion data for the pancreas (green) and spleen (yellow) of the same rat. (D) Schematic diagram of linearized perfusion data indicating wash-in rate, wash-in area, and peak enhancement. (E) Measured wash-in area, wash-in rate, and peak enhancement of pIC+KRV treated and control rats at selected time points; Day 1 (red), Day 6 (blue) and Day 11 (purple) post-induction and age-matched control rats (green). n = 3, error bars represent SE; *p<0.05 and ***p<0.001 for Day 1 pIC+KRV treated rats versus all other groups.
Fig 6.
Vessel resistance in the superior mesenteric artery is significantly reduced at later stages following diabetes induction.
Ultrasound imaging was first used to locate the superior mesenteric artery (SMA) and Pulsed Wave Doppler mode was then used to measure its peak systolic and end diastolic velocity. (A) Representative ultrasound image showing the location of the SMA, aorta, and kidney. (B) Representative ultrasound image using Pulsed Wave Doppler mode to measure peak systolic and end diastolic velocity of the SMA for calculation of the Resistivity Index. The lower green and yellow lines show vital signs for heart rate and breathing cycle, respectively, from a control rat. (C) The calculated Resistivity Index of the SMA of pIC+KRV treated and control rats at the times indicated. n = 3, error bars show SE; *p<0.05 for Day 6 and Day 11 pIC+KRV treated rats versus Day 1 pIC+KRV treated rats and age-matched control rats.