P68: Laser Doppler perfusion imaging for non-invasive assessment of vascular responses in living mice

Abstract

Background Methods for the assessment of vascular functions in mice are largely limited to ex vivo organ bath methods particularly the aortic ring assay. We aimed at establishing an easy to perform, sensitive, and valid method for the non-invasive measurement of vascular responses to pharmacological and physiological stimuli in vivo using laser Doppler perfusion imaging (LDPI). Methods Male C57Bl/6, 8–10 week old mice were anesthetized with isoflurane and tissue perfusion of the hindlimb was assessed by LDPI (Perimed, Stockholm, Sweden). The changes in tissue perfusion in response to vasodilators and vasoconstrictors was assessed by acute intraperitoneal (i.p) application of nitroglycerin, intravenous (i.v.) application of acethylcholine, and i.p application of epinephrine. Reactive hyperemic blood flow was induced by occluding the vessels of the hindlimb using an inflatable cuff until the perfusion signal reached the zero level. After 1–5 min of ischemia the cuff pressure was released. We determined intra-individual variability by measuring the same mouse on three consecutive days ( n = 4) and inter-individual variability by comparing the coefficient of variation (CV) of 11 mice. The effect of acute and chronic inhibition of nitric oxide synthase (NOS) or cyclooxygenase (COX) on the hyperemic response was achieved by administration of l -NNA or indometacin i.p or for 6 days in drinking water ad libitum, respectively. Results Application of nitroglycerin increased the perfusion of the hindlimb within approximately 100 s by 127% (191 ± 36 vs. 435 ± 23 perfusion units, PU), while epinephrine (209 ± 59 vs. 90 ± 18 PU:) resulted in a maximum decrease of 57% of the mean perfusion ( n = 5). I.v. administration of adenosine elevated the perfusion by 41% (224 ± 34 vs. 316 ± 43 PU). Reactive hyperemia progressively increased after 1, 3 and 5 min ischemia with the maximal response achieved after 5 min ischemia. Time to maximum reperfusion and the ratio of maximum/baseline perfusion values (perfusion reserve) showed the smallest variation, while the area under the perfusion curve exhibited a CV of 40–50%. Generally, intra-individual variability was lower as compared to inter-individual variability. Chronic oral treatment with l -NNA significantly prolonged the time to maximal perfusion (36 ± 5 vs. 77 ± 18 s), while acute NOS inhibition ( l -NNA i.P) reduced the perfusion reserve (2.5 ± 0.1 vs. 1.8 ± 0.0). In addition, chronic inhibition of COX resulted in a 16 s longer reperfusion (48 ± 5 vs. 64 ± 5 s) but had no effect on the other parameters. Conclusion Taken together, LDPI can be used as a valid and reproducible method for measuring vascular responses to physiological and pharmacological stimuli in the hindlimb of living mice. This may be a valuable experimental model that is similar to methodologies applied in humans enabling a translation of basic science findings. Disclosure Nothing to disclose.