Figure 1.
Luciferase expression is restricted to the pancreas.
A) An example of BLI on ob/ob-luc mice. The animals of the same age were injected i.p. with D-luciferin and imaged 5 min later. Signal is present in the midsection of the animals only. B) Biodistribution of luciferase in the organs of ob/ob-luc mice. Three animals with similar intensities in the midsection were dissected and the organs were imaged with an IVIS Spectrum. ROI analysis was performed on the organs. The background bar is highlighted to show all organs except pancreas are at background levels while pancreas is significantly higher than other organs (*p<0.001, One way ANOVA). C) Zoomed in BLI of a pancreas. D) The pancreas was sectioned and stained with anti-insulin antibody. Both BLI and insulin staining have similar punctate patterns.
Table 1.
Coefficient of Variation for Area Under the Curve measurements of i.p. vs. i.v. administration of D-luciferin in ob/ob-luc mice.
Figure 2.
Longitudinal images of B6 albino MIP-luc lean (lean controls) and B6 albino MIP-luc ob/ob (ob/ob-luc) mice.
A) Representative BLI of ob/ob-luc and lean controls over time. In ob/ob-luc mice the area of signal also increases, which could indicate an increase in the number of islets with age. However, the scatter of photons by adipose tissue may also contribute to the size of the detected area. Note: lean control mice are on a separate color scale since ob/ob-luc mice have higher luciferase expression B) Results of ROI analysis was performed on the pancreas signal of both ob/ob-luc (closed symbols) and lean control mice (open symbols) (N = 13 each). The ROI was drawn over the entire signal and presented as total flux (p/s). In ob/ob-luc mice the signal increases with age while in lean controls it remains unchanged.
Figure 3.
Characterization of ob/ob-luc mice.
A) Weight increases significantly with age in ob/ob-luc mice consistent with the original ob/ob mouse model on the B6 black background. As early as week 8 the ob/ob-luc mice are 2.5 times the weight of their lean control counterparts. Blood was taken from fasting mice following imaging to measure B) glucose and C) insulin. Glucose levels in ob/ob-luc mice (closed symbols) are significantly higher than lean controls (open symbols) at week 8 (p<0.001, t-test). As the ob/ob-luc mice age the glucose levels decrease and reach similar levels as lean controls by week 45. Insulin levels in ob/ob-luc mice increase with age while insulin levels in lean mice remain stable with age. Both glucose and insulin levels follow similar trends as normal ob/ob mice, indicating that the additional genetic manipulation (albino and MIP-luc) did not affect the physical characteristics of the ob/ob model. D) There is a good correlation (R2 = 0.79) between plasma insulin levels and in vivo BLI measurements.
Figure 4.
Correlation of ex vivo bioluminescence with quantitative histology.
A) Total number of β-cells was counted from sections of pancreas from ob/ob-luc mice (N = 3–8/group). Mice were grouped to minimize variation since individual mice reach disease stages at different time points. B) Total bioluminescence was measured from ex vivo images of pancreata from ob/ob-luc mice. When the data are grouped similar to β-cell number there is an upwards trend in β-cell number as the mice age. (The horizontal lines in figures A and B indicate a statistically significant difference between the two groups, *p<0.001, One way ANOVA) C) There is a strong correlation between the number of β-cells counted by histology and ex vivo BLI measurements.
Figure 5.
Insulin measurements on individual islets confirms in vivo BLI and insulin measurements.
A) Bioluminescence imaging of islets isolated from ob/ob luciferase mice at 10, 22, 40, and 64 weeks of age displayed increased bioluminescence with increasing age up to 40 weeks of age, followed by decreased BLI at 64 weeks of age. B) Isolated islets displayed increased total insulin content with increasing age up to 40 weeks of age. Insulin content of islets from 64 week old mice was not significantly different than at 40 weeks. C) Isolated ob/ob-luc islets displayed insulin release in response to insulin secretagogues. The response from 64 week old islets is represented by open symbols to distinguish it from the other groups. High glucose (16.7 mM) and IBMX induced higher insulin release in islets from 40 week old mice and lower insulin release in islets from 64 week old mice (*p = 0.015, One way ANOVA).