Non-invasive real-time pulsed Doppler assessment of blood flow in mouse ophthalmic artery

Figure 1. Setup of non-invasive Doppler OphA blood flow velocity measurements at mouse orbital opening An anesthetized mouse is placed in a prone position on the ECG board. The Doppler probe sensor tip is at the temporal canthus (left and center). Doppler in-phase (I), quadrature (Q), and ECG signals are acquired, processed, displayed, and recorded in real-time (middle). Doppler velocity signal waveforms with major parameters are acquired for analysis (right). DVFS, Doppler flow velocity system; US, ultrasound; ECG, electrocardiogram; FV, flow velocity.

Figure 2. Defining the ultrasound probe signal trajectory by microCT (A and B) Mouse skull images with the fiduciary needle in place (A) and after removal (B). (C) MicroCT imaging of a mouse following transcardiac perfusion with Vascupaint to visualize the arterial vasculature. The white dashed circle indicates the region targeted by the ultrasound probe. OphA indicates the ophthalmic artery is emerging from the optic canal. (D–F) In vivo microCT vascular tracing of the OphA from its origin off the proximal internal carotid artery (ICA) to its emergence into the orbit. (D) View into the orbit from outside the skull. (E) View from superior aspect showing OphA path, circle of Willis, and major branches. (F) View from inferior aspect showing the origin of the OphA from the ICA. The OphA is color coded in green and the ICA and branches of the circle of Willis are in blue. CCA, common carotid artery.

Figure 3. Targeted artery (OphA) originates as a branch off the ICA (A) Experimental setup for sequential ECA and ICA branch occlusion. A surgically prepared mouse on the heated board with continuous ECG monitoring, the Doppler probe, and the dissected area are shown. (B) Magnified view of the neck region prepared for occlusion. CCA, common carotid artery; ICA, internal carotid artery; ECA, external carotid artery. (C) Schematic representation of anatomical features in close proximity to CCA and ICA/ECA branching site (adapted from Liu et al.87). (D) Changes of the Doppler signal during sequential occlusion of the blood flow in left ECA and ICA. A representative of 6 independent sequential occlusions from 3 individual mice is shown.

Figure 4. Hemodynamic response to SNP bolus (A) OphA blood flow velocity (BFV; cm/s) waveforms at baseline (BL), 3 min post-injection (3 min PI), and 15 min PI. (B) Dynamics of OphA PFV changes in anesthetized mice after 1 mg/kg SNP injection, plotted as a percentage of baseline. Data are mean ± SD for 3-month-old male (M; n = 5), female (F; n = 5), and control (C; n = 10) mice. Control mice were injected with PBS. ∗p < 0.05 versus females (two-way ANOVA and Bonferroni multi-comparison test). (C) Dynamic changes in systemic blood pressure following SNP injection. Data are mean ± SD for 3 male and 3 female mice.

Figure 5. OphA PVF changes during sequential surgical ligation of common carotid arteries (A) OphA blood flow velocity waveforms showing changes in PFV at baseline, carotid artery occlusion, and after re-opening of left and right carotid arteries. The sequence: BL (baseline), RCc (right common carotid closed), LCc (left common carotid closed), LCo (left common carotid re-opened), and RCo (right common carotid re-opened). (B) PFV changes during surgical manipulations presented as a percentage of baseline. Data are mean ± SD from 6-month-old BALB/C males (n = 5).