A method for noninvasive longitudinal measurements of [Ca²⁺] in arterioles of hypertensive optical biosensor mice

Abstract

Mauban JR, Fairfax ST, Rizzo MA, Zhang J, Wier WG. A method for noninvasive longitudinal measurements of [Ca 2ϩ ] in arterioles of hypertensive optical biosensor mice. Am J Physiol Heart Circ Physiol 307: H173-H181, 2014. First published May 23, 2014 doi:10.1152/ajpheart.00182.2014.-We used two-photon (2-p) Förster resonance energy transfer (FRET) microscopy to provide serial, noninvasive measurements of [Ca 2ϩ ] in arterioles of living "biosensor" mice. These express a genetically encoded Ca 2ϩ indicator (GECI), either FRET-based exMLCK or intensity-based GCaMP2. The FRET ratios, Rmin and Rmax, required for in vivo Ca 2ϩ calibration of exMLCK were obtained in isolated arteries. For in vivo experiments, mice were anesthetized (1.5% isoflurane), and arterioles within a depilated ear were visualized through the intact skin (i.e., noninvasively), by 2-p excitation of exMLCK (at 820 nm) or GCaMP2 (at 920 nm). Spontaneous or agonist-evoked [Ca 2ϩ ] transients in arteriolar smooth muscle cells were imaged (at 2 Hz) with both exMLCK and GCaMP2. To examine changes in arteriolar [Ca 2ϩ ] that might accompany hypertension, five exMLCK mice were implanted with telemetric blood pressure transducers and osmotic minipumps containing ANG II (350 ng·kg Ϫ1 ·min Ϫ1 ) and fed a high (6%)-salt diet for 9 days. [Ca 2ϩ ] was measured every other day in five smooth muscle cells of two to three arterioles in each animal. Prior to ANG II/salt, [Ca 2ϩ ] was 246 Ϯ 42 nM. [Ca 2ϩ ] increased transiently to 599 nM on day 2 after beginning ANG II/salt, then remained elevated at 331 Ϯ 42 nM for 4 more days, before returning to 265 Ϯ 47 nM 6 days after removal of ANG II/salt. In summary, two-photon excitation of exMLCK and GCaMP2 provides a method for noninvasive, longitudinal quantification of [Ca 2ϩ ] dynamics and vascular structure in individual arterioles of a particular animal over an extended period of time, a capability that should enhance future studies of hypertension and vascular function.calcium; FRET; hypertension; two photon; vascular STUDIES of experimental hypertension in animals are confronted with several fundamental technical difficulties. Determining how changes in arterial [Ca 2ϩ ] might be involved in experimental hypertension is particularly challenging because measurements of [Ca 2ϩ ] and artery contractile activation will change immediately upon removal of an artery for study. The processes controlling [Ca 2ϩ

Imaging sympathetic neurogenic Ca 2+ signaling in blood vessels

“…The development of optical biosensor mice has been critical to the advancement of in vivo imaging studies of SNS control of arterial function (Zhang et al , 2010, Mauban et al , 2013, Zacharia et al , 2013, Mauban et al , 2014, Ji et al ., 2004). These mice express genetically engineered Ca 2+ sensor molecules specifically in smooth muscle.…”

“…The relationship between free [Ca 2+ ] and exMLCK FRET ratio, R, as measured in α-toxin permeabilized mesenteric small arteries, is well fitted by the Hill equation, with an EC 50 (K A ) of 0.892 μM (pCa, 6.05) and a Hill Coefficient ( n ) of 1.4 (Wang et al, 2013). Methods for determination of intrinsic fluorescence, spectral overlap and other considerations for quantitative imaging of [Ca 2+ ] using exMLCK FRET ratio measurements under these conditions were described previously (Wier et al , 2008, Zhang et al , 2010, Mauban et al , 2014)…”

“…Further immobilization or restraint of the animal may also be required. In our recent experiments (Fairfax et al , 2014), the animals’ heads were fixed in position by attaching the threaded bar to an anchored stage, their bodies were immobilized inside a custom-made conical tube, and their depilated ears were positioned on a flat horizontal silicone platform as previously detailed (Mauban et al , 2014). This arrangement immobilized the ear, without directly pinning it, and provided a convenient configuration for use of a dipping objective lens (20×, 1.0 NA).…”

“…The gains of the two detector channels were held constant for all experiments at levels optimizing exMLCK fluorescence. The configuration settings of the detectors were reproduced during calibration experiments, which determined the maximum and minimum FRET ratios obtainable from our microscope (Mauban et al , 2014). …”

“…In such studies, tonic SNA to arterioles has been associated with vasomotion and a particular kind of Ca 2+ signaling. A point that will be emphasized here is that the use of such techniques permit longitudinal studies of vascular function (Mauban et al , 2014, Fairfax et al , 2014). Briefly, the use of non-invasive, non-perturbing experimental techniques in optical biosensor mice means that an individual animal can be studied over time, including the induction, development and reversal of an experimental condition or disease model.…”