Spectral imaging reveals microvessel physiology and function from anastomoses to thromboses

Abstract

Abnormal microvascular physiology and function is common in many diseases. Numerous pathologies include hypervascularity, aberrant angiogenesis, or abnormal vascular remodeling among the characteristic features of the disease, and quantitative imaging and measurement of microvessel function can be important to increase understanding of these diseases. Several optical techniques are useful for direct imaging of microvascular function. Spectral imaging is one such technique that can be used to assess microvascular oxygen transport function with high spatial and temporal resolution in microvessel networks through measurements of hemoglobin saturation. We highlight novel observation made with our intravital microscopy spectral imaging system employed with mouse dorsal skin-fold window chambers for imaging hemoglobin saturation in microvessel networks. Specifically, we image acute oxygenation fluctuations in a tumor microvessel network, the development of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia, and the formation of spontaneous and induced microvascular thromboses and occlusions.

Figure 1

Transmitted light image of a 4TO7 tumor and adjacent normal tissue. ROIs for HbSat measurements in Figs. 234 are marked with the image. ROI 1, venule; ROI 2, arteriole; ROI 3, venule; ROI 4, arteriole; ROI 5, tumor feeding arteriole; ROI 6, tumor feeding arteriole; ROIs 7 to 9, tumor microvessels.

Figure 2

HbSat in ROIs 1 to 6 in Fig. 1. Measurements were acquired every 20 seconds for 43 minutes. Data points are omitted for clarity.

Figure 3

HbSat in ROIs 7 9 in Fig. 1. Measurements were acquired every 20 s for 43 min. Data points are omitted for clarity.

Figure 4

Plot of vessel with ROI 7 HbSat measurements (abscissas) versus vessels with ROIs 3 to 6 (ordinates) in Fig. 1 for the time points over the 43-min imaging session with data sets acquired every 20 s. The specific ROI being compared to ROI 7 is indicated in each graph. A line was fit to the data to gauge the correlation between the oxygenation in the vessels and R² values of the linear fit are indicated in the figure.

Figure 5

AVM development in a conditional Alk1 deletion mouse (ROSA26^CreER∕Alk1^2f∕−) stimulated by a wound, where A, arteriole; V, venule; and W, wound. The left column shows transmitted light images and the right column shows HbSat images. Region where AVMs formed is indicated in the day 5 and day 9 images. At day 5, the AVM connections were not obvious but clear changes in venule oxygenation could be seen due to the AVMs. The arrowheads indicating the arteriole and venule also indicate regions of interest where HbSat measurements were obtained.

Figure 6

Wound healing in a control mouse: A, arteriole; V, venule; and W, wound. The left column shows transmitted light brightfield images and the right column shows HbSat images. The arrowheads indicating the arteriole and venule also indicate regions of interest where HbSat measurements were obtained. Unlike the conditional Alk1 deletion mouse in Fig. 5, no AVMs formed during wound healing.

Figure 7

Brightfield and HbSat images are shown before and 21 minutes after topical application of FeCl₃ to induce thrombus formation. The red dotted line in the top left image indicates where the border of the paper soaked with FeCl₃ was relative to the image area. The yellow box indicates the region shown in the images in Fig. 8 at higher magnification. (Color online only.)

Figure 8

Brightfield and HbSat time sequence images of thrombus development for the region indicated by the yellow box in Fig. 7. The yellow outlined area on the venule in the top left image indicates the ROI used for the HbSat measurements shown in Fig. 9. The arrow in the brightfield image at the 8-min time point indicates a forming thrombus. (Color online only.)

Figure 9

Plot of the HbSat in the ROI indicated in Fig. 8 at various time points during thrombus development. The data points in the figure are given as the median±interquartile range for the ROI.

Figure 10

Tumor microvessel network in which a spontaneous RBC occlusion formed during imaging. Within the highlighted region is the vessel with the occlusion. The vessel is marked with several ROIs that were used to determine the apparent location of the occlusion front as it moved during the imaging session. The location of the occlusion front at the beginning and end of the imaging session are indicated in the figure. The highlighted region with the occluded vessel is shown in more detail in Fig. 11.

Figure 11

Detail of highlighted region in Fig. 10 showing the progression of the RBC occlusion front during the imaging session (arrow). The RBC occlusion front progressed counter to the direction of blood flow in the vessel. The occlusion front was chosen by identifying an area within the HbSat gradient in the occlusion vicinity where the HbSat decreased to a level less than 5%. The HbSat values of several ROIs on the apparent occlusion front at different time points are indicated in the figure.

Figure 12

Hemoglobin saturation maps of the tumor microvessel network in Fig. 10 at two different time points. The images were taken at (a) 5 and (b) 60 min into the imaging session. The plug (P) is indicated in (a). Note the sharp change in vessel oxygenation at the plug relative the perfused region upstream of the occlusion. A steep vessel oxygenation gradient (G) is seen in near the plug as the plug advances in the vessel in (b).