Ex Vivo Imaging of Ultrasound-Stimulated Metabolic Activity in Rat Pancreatic Slices

Abstract

Ultrasound has previously been reported to produce a reversible stimulatory effect in cultured rat beta cells. Here, we quantified and assessed dynamic metabolic changes in an in situ pancreatic slice model evoked by ultrasound application. After plating, pancreas slices were imaged using a confocal microscope at 488 and 633 nm to image lipodamine dehydrogenase (Lip-DH) autofluorescence and a far red fluorescence, respectively. Ultrasound was applied at intensities of 0.5 and 1 W/cm² at both 800 kHz and 1 MHz. Additionally, 800 kHz at 1 W/cm² was applied in a pulsing scheme. No ultrasound (control) and glucose application experiments were performed. A difference in fluorescence signal before and after treatment application was the metric for analysis. Comparison of experimental groups using far red fluorescence revealed significant differences between all experimental groups and control in the islet (p < 0.05) and between all ultrasound experimental groups and control (p < 0.05) in pancreatic exocrine tissue. However, this difference in response between control and glucose did not exist in the exocrine tissue. We also observed using Lip-DH autofluorescence that glucose produces a significantly increased metabolic response in islet tissue compared with exocrine tissue (p < 0.05). Pulsed ultrasound appeared to increase metabolic activity in the pancreatic slice in a more consistent manner compared with continuous ultrasound application. Our results indicate that therapeutic ultrasound may have a stimulatory metabolic effect on the pancreatic islets similar to that of glucose.

Keywords: Fluorescence imaging; Pancreas; Therapeutic ultrasound; Type 2 diabetes.

Figure 1.

Illustrated process of perfusing the pancreas with agarose. The inferior part of the liver is flipped over the superior end revealing the pancreas and duodenum. The duodenum is clamped and agar delivered to the pancreas through the common bile duct. The pancreas is filled with agar by retrograde perfusion through the pancreatic duct.

Figure 2.

(a) A section of the pancreas perfused with agar and embedded in agar. (b) Slicing of the agar perfused and embedded pancreas on a vibrating microtome. (c) Confocal microscope with XYZ positioner mounted to optical table (white arrow). (d) Ultrasound transducer (white arrow) inside imaging well during time series image capture. (e) Bright field, white arrow indicates the edge of the 14 mm glass bottom micro well (f) 488 nm and (g) 633 nm light application. (h) bright field image (i) bright field overlaid with 488 nm imaging (j) bright field overlaid with 633 nm imaging (k) combination image of bright field with 488 nm and 633 nm imaging. (h) (i) (j) & (k) black arrow indicates islet. Scale bar 100 μm.

Figure 3.

The custom slice spatula used to transport pancreas slices. (a) The pancreas tissue in the slice (black arrow) can sometimes be sparse and held together largely by agar. (b) The spatula with no slice.

Figure 4.

Example of motion detection software. White arrow indicates where motion can be seen as a separation of the islet/exocrine tissue boundary from the red landmark point. Scale bar 100 μm.

Figure 5.

PZFlex Model used to simulate ultrasound application. The 14mm micro well in this geometry contains the thin pancreas slice (red) along with a layer of agar above it. The large white box is the submerged transducer.

Figure 6.

(a) & (b) Representative pressure and temperature maps with a red rectangle indicating the area of the pancreas slice. This is a zoomed in geometry of the simulation geometry in Figure 5. (c) & (d) Pressure and temperature plots across a pancreas slice.

Figure 7.

Temperature measurements taken from inside imaging well with a thermocouple. The pulsing scheme significantly lowers the amount of heating during a three minute application period.

Figure 8.

ANOVA with Tukey-Kramer significance test for all experimental groups separated by tissue type and color channel. Color channels are 488 (a) & (b) and 633 (c) & (d). Tissue type is islet (blue) and exocrine (red). Color channels are 488 nm excitation which indicates changes in levels of Lip-DH and 633 nm for a far red fluorescence signal. Significant differences exist between control and all other parameters in the FR fluorescence for pancreatic islets (*p<0.05) as well as exocrine pancreas tissue except for the difference between glucose application and control in exocrine tissue. This may indicate that ultrasound and glucose both have an effect when compared to control, however the effect of glucose may be islet specific while ultrasound may act bilaterally.

Figure 9.

Paired t-test results of the various experimental groups. There is a significant difference (*p<0.05) in the 488 nm color channel for the glucose experimental group between islet and exocrine tissue.