Bedding as a variable affecting fasting blood glucose and vascular physiology in mice.

Rodent husbandry requires careful consideration of environmental factors that may impact colony performance and subsequent physiological studies. Of note, recent reports have suggested corncob bedding may affect a broad range of organ systems. As corncob bedding may contain digestible hemicelluloses, trace sugars, and fiber, we hypothesized that corncob bedding impacts overnight fasting blood glucose and murine vascular function. Here, we compared mice housed on corncob bedding, which were then fasted overnight on either corncob or ALPHA-dri bedding, a virgin paper pulp cellulose alternative. Male and female mice were used from two noninduced, endothelial-specific conditional knockout strains [Cadherin 5-cre/ERT2, floxed hemoglobin-α1 (Hba1fl/fl) or Cadherin 5-cre/ERT2, floxed cytochrome-B5 reductase 3 (CyB5R3fl/fl)] on a C57BL/6J genetic background. After fasting overnight, initial fasting blood glucose was measured, and mice were anesthetized with isoflurane for measurement of blood perfusion via laser speckle contrast analysis using a PeriMed PeriCam PSI NR system. After a 15-min equilibration, the mice were injected intraperitoneally with the α1-adrenergic receptor agonist, phenylephrine (5 mg/kg), or saline, and monitored for changes in blood perfusion. After a 15-min response period, blood glucose was remeasured postprocedure. In both strains, mice fasted on corncob bedding had higher blood glucose than the pulp cellulose group. In the CyB5R3fl/fl strain, mice housed on corncob bedding displayed a significant reduction in phenylephrine-mediated change in perfusion. In the Hba1fl/fl strain, phenylephrine-induced change in perfusion was not different in the corncob group. This work suggests that corncob bedding, in part due to its ingestion by mice, could impact vascular measurements and fasting blood glucose. To promote scientific rigor and improve reproducibility, bedding type should be routinely included in published methods.NEW & NOTEWORTHY This study demonstrates real-time measurement of changes in perfusion to pharmacological treatment using laser speckle contrast analysis. Furthermore, this investigation revealed that fasting mice overnight on corncob bedding has differential effects on vascular function and that there was increased fasting blood glucose in mice fasted on corncob bedding compared with paper pulp cellulose bedding. This highlights the impact that bedding type can have on outcomes in vascular and metabolic research and reinforces the need for thorough and robust reporting of animal husbandry practices.

Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum.

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases the risk for neurodegeneration. Among various cells that participate in BBB and NVU function, calcium signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with altered calcium signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed GCaMP6f in DLS astrocytes of young (3-4 months), middle-aged (12-15 months) and aging (20-30 months) mice. Compared to endfeet in young mice, DLS endfeet in aging mice demonstrated decreased calreticulin expression, and alterations to both spontaneous membrane-associated and mitochondrial calcium signals. While young mice required both extracellular and endoplasmic reticulum calcium sources for endfoot signals, middle-aged and aging mice showed heavy dependence on endoplasmic reticulum calcium. Thus, astrocytic endfeet show significant changes in calcium buffering and sources throughout the lifespan, which is important for understanding mechanisms by which aging impairs the BBB and NVU.

Microvascular dysfunction and kidney disease: Challenges and opportunities?

Kidneys are highly vascular organs that despite their relatively small size receive 20% of the cardiac output. The highly intricate, delicately organized structure of renal microcirculation is essential to enable renal function and glomerular filtration rate through the local modulation of renal blood flow and intraglomerular pressure. Not surprisingly, the dysregulation of blood flow within the microvessels (abnormal vasoreactivity), fibrosis driven by disordered vascular-renal cross talk, or the loss of renal microvasculature (rarefaction) is associated with kidney disease. In addition, kidney disease can cause microcirculatory dysfunction in distant organs such as the heart and brain, mediated by mechanisms that remain to be elucidated. The objective of this review is to highlight the role of renal microvasculature in kidney disease. The overview will outline the impetus to study renal microvasculature, the bidirectional relationship between kidney disease and microvascular dysfunction, the key pathways driving microvascular diseases such as vasoreactivity, the cell dynamics coordinating fibrosis, and vessel rarefaction. Finally, we will also briefly highlight new therapies targeting the renal microvasculature to improve renal function.

Comparison of Adrenergic and Purinergic Receptor Contributions to Vasomotor Responses in Mesenteric Arteries of C57BL/6J Mice and Wistar Rats.

INTRODUCTION: The sympathetic nervous system can modulate arteriolar tone through release of adenosine triphosphate and norepinephrine, which bind to purinergic and adrenergic receptors (ARs), respectively. The expression pattern of these receptors, as well as the composition of neurotransmitters released from perivascular nerves (PVNs), can vary both in organ systems within and across species, such as mice and rats. OBJECTIVE: This study explores the function of α1A subtypes in mouse and rat third-order mesenteric arteries and investigates PVN-mediated vasoconstriction to identify which neurotransmitters are released from sympathetic PVNs. METHODS: Third-order mesenteric arteries from male C57BL/6J mice and Wistar rats were isolated and mounted on a wire myograph for functional assessment. Arteries were exposed to phenylephrine (PE) and then incubated with either α1A antagonist RS100329 (RS) or α1D antagonist BMY7378, before reexposure to PE. Electrical field stimulation was performed by passing current through platinum electrodes positioned adjacent to arteries in the absence and presence of a nonspecific alpha AR blocker phentolamine and/or P2X1-specific purinergic receptor blocker NF449. RESULTS: Inhibition of α1 ARs by RS revealed that PE-induced vasoconstriction is primarily mediated through α1A and that the contribution of the α1A AR is greater in rats than in mice. In the mouse model, sympathetic nerve-mediated vasoconstriction is mediated by both ARs and purinergic receptors, whereas in rats, vasoconstriction appeared to only be mediated by ARs and a nonpurinergic neurotransmitter. Further, neither model demonstrated that α1D ARs play a significant role in PE-mediated vasoconstriction. CONCLUSIONS: The mesenteric arteries of male C57BL/6J mice and Wistar rats have subtle differences in the signaling mechanisms used to mediate vasoconstriction. As signaling pathways in humans under physiological and pathophysiological conditions become better defined, the current study may inform animal model selection for preclinical studies.

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

Morphological and pharmacological characterization of the porcine popliteal artery: A novel model for study of lower limb arterial disease.

OBJECTIVE: This study was undertaken to characterize structural and pharmacological properties of the pig popliteal artery in order to develop a novel system for the examination of lower limb blood flow regulation in a variety of cardiovascular pathologies, such as diabetes-induced peripheral artery disease. METHODS: Popliteal arteries were isolated from streptozocin-induced diabetic pigs or age-matched saline-injected control pigs for morphological study using transmission electron microscopy and for examination of vasoreactivity to pharmacological agents using wire myography. RESULTS: Transmission electron microscopy of the porcine popliteal artery wall revealed the presence of endothelial cell-smooth muscle cell interactions (myoendothelial junctions) and smooth muscle cell-smooth muscle cell interactions, for which we have coined the term "myo-myo junctions." These myo-myo junctions were shown to feature plaques indicative of connexin expression. Further, the pig popliteal artery was highly responsive to a variety of vasoconstrictors including norepinephrine, phenylephrine, and U46619, and vasodilators including acetylcholine, adenosine 5'-[ß-thio] diphosphate, and bradykinin. Finally, 2 weeks after streptozocin-induced diabetes, the normalized vasoconstriction of the pig popliteal artery to norepinephrine was unaltered compared to control. CONCLUSIONS: The pig popliteal artery displays structural and pharmacological properties that might prove useful in future studies of diabetes-associated peripheral artery disease and other lower limb cardiovascular diseases.

VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells.

KEY POINTS: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca2+ release and subsequent activation of intermediate conductance Ca2+ -activated K+ (IKCa ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca2+ responses and the EDH-vasodilatation mediated by IKCa activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IKCa channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases. ABSTRACT: Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca2+ release and the associated EDH vasodilatation mediated by intermediate conductance Ca2+ -activated K+ (IKCa ) channels. Importantly, VEGF-A had no direct effect against IKCa channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP3 ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP3 R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca2+ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles.

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca2+ signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca2+ or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. We provide evidence that Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca2+ signaling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca2+ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca2+-activated K+ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow.

Scaffolding builds to reduce blood pressure.

Endothelial cells provide vasodilator signals to reduce blood pressure. In the small resistance arteries and arterioles, which determine the distribution and pressure of blood, the major signal is hyperpolarization reflecting the endothelial activity of calcium-activated potassium channels (KCa). In this issue of Science Signaling, Sonkusare et al. report that the scaffold protein AKAP150 is required for the kinase PKC and the calcium channel TRPV4 to enable receptor-mediated relaxation signaling. This scaffold enhances TRPV4 gating cooperativity and markedly amplifies the Ca(2+) signal, which ultimately activates (mainly) IKCa channels. Normally restricted to tiny endothelial projections, AKAP150 localization and associated signaling is disrupted in a model of hypertension, thereby diminishing hyperpolarization and vasodilation.

ß1-integrin is essential for vasoregulation and smooth muscle survival in vivo.

OBJECTIVE: Integrins contribute to vascular morphogenesis through regulation of adhesion and assembly of the extracellular matrix. However, the role of ß1-integrin in the mature vascular wall is less clear. APPROACH AND RESULTS: We sought to determine the function of ß1-integrin in mature smooth muscle cells in vivo using a loss of function approach by crossing a tamoxifen-inducible sm22αCre line to a floxed ß1-integrin transgenic line. Adult mice lacking smooth muscle ß1-integrin survived only 10 weeks post induction. The deletion of ß1-integrin resulted in profound loss of vasomotor control. Histological analysis revealed progressive fibrosis in arteries with associated apoptosis of smooth muscle cells, which was not rescued by adventitial stem cells. Smooth muscle cell apoptosis was detected in arteries with dead cells replaced primarily by collagen. Despite the catastrophic effects on vascular smooth muscle, the deleted visceral smooth muscle remained viable with the exception of a short portion of the colon, indicating that vascular but not visceral smooth muscle is particularly sensitive to changes in ß1-integrin. CONCLUSIONS: This study reveals an essential function of ß1-integrin in the maintenance of vasomotor control and highlights a critical role for ß1-integrin in vascular, but not visceral, smooth muscle survival.

MGRN1-dependent pigment-type switching requires its ubiquitination activity but not its interaction with TSG101 or NEDD4.

Mice lacking the E3 ubiquitin ligase mahogunin ring finger-1 (MGRN1) have a pleiotropic phenotype that includes spongiform neurodegeneration, embryonic patterning defects, and dark fur due to a defect in pigment-type switching. The only MGRN1 ubiquitination target identified to date is tumor susceptibility gene 101 (TSG101), a component of the endosomal trafficking machinery. Here, we show that MGRN1 also interacts with but does not ubiquitinate NEDD4, a HECT-domain ubiquitin ligase involved in endosomal trafficking. Using transgenesis in mice, we demonstrate that pigment-type switching likely requires MGRN1's ubiquitin ligase activity but not its ability to bind TSG101 or NEDD4. This indicates that MGRN1-dependent ubiquitination of an as-yet unidentified target protein is required for agouti-mediated melanocortin signaling.

Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone.

Endothelial cell (EC) Ca(2+)-activated K channels (SK(Ca) and IK(Ca) channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK(Ca) channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca(2+) events (Ca(2+) puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (~2 min(-1)) inositol 1,4,5-trisphosphate receptor-based Ca(2+) events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca(2+) events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca(2+) increases, which on occasion led to intracellular Ca(2+) waves. The concurrent vasodilation associated with increases in Ca(2+) event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK(Ca) channel blocker), but not by apamin (SK(Ca) channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca(2+) event frequency; at low pressures the consequence is activation of EC IK(Ca) channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.

The mouse cremaster muscle preparation for intravital imaging of the microcirculation.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation. The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem. The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light. However curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope. While shown to be a valuable preparation to study the physiology of the microcirculation in rats and hamsters, the cremaster muscle in mice has proven particularly useful in dissecting cellular pathways involved in regulating microvascular function and real-time imaging of intercellular signaling. The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal. It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a genetically encoded fluorescent calcium indicator molecule. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of calcium signaling in the arteriolar endothelium.

Microiontophoresis and micromanipulation for intravital fluorescence imaging of the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission by delivering neurotransmitters and neuropeptides onto neurons reproducibly. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels. A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be ˜ 1 µm or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette. Fabrication of micropipettes is facilitated with sophisticated electronic pullers. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). The present protocol demonstrates microiontophoresis of acetylcholine (ACh(+) Cl(-)) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40(BAC)-GCaMP2 transgenic mice enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Visualizing calcium responses to acetylcholine convection along endothelium of arteriolar networks in Cx40BAC-GCaMP2 transgenic mice.

Acetylcholine evokes endothelium-dependent vasodilation subsequent to a rise in intracellular calcium. Despite widespread application in human and animal studies, calcium responses to intravascular ACh have not been visualized in vivo. Microiontophoresis of ACh in tissue adjacent to an arteriole activates abluminal muscarinic receptors on endothelial cells within a "local" region of diffusion, but it is unknown whether ACh released in such fashion gains access to the flow stream resulting in further actions downstream. To test this hypothesis and provide new insight into calcium signaling in vivo, we studied the cremaster muscle microcirculation of anesthetized male Cx40(BAC)-GCaMP2 transgenic mice (n = 22; 5-9 mo; 33 ± 1 g) expressing the fluorescent calcium sensor GCaMP2 selectively in arteriolar endothelial cells. Submaximal ACh stimuli were delivered using microiontophoresis (1-µm pipette tip, 500 nA). With stimulus duration <500 ms or with the micropipette positioned within one vessel diameter (∼30 µm) away from an arteriole, endothelial cell calcium fluorescence was restricted to the region of ACh diffusion (<200 µm). In contrast, with the micropipette tip positioned immediately adjacent to an arteriole or within its lumen, calcium fluorescence encompassed entire networks downstream. The velocity of downstream calcium signaling in response to ACh increased with centerline velocity of fluorescent tracer microbeads (r(2) > 0.99; range: <1 mm/s to >10 mm/s). Diverting arteriolar blood flow into a side branch redirected downstream fluorescence responses to ACh; occluding flow abolished responses. Blocking luminal muscarinic receptors (intravascular glycopyrrolate; 6 µg/kg) inhibited downstream responses reversibly. Through visualizing the actions of a "local" ACh stimulus on endothelial cell calcium fluorescence in vivo, the present findings illustrate that transmural diffusion and convection of an agonist can activate entire networks of arteriolar endothelial cells concomitant with its delivery in the flow stream.

Intravital macrozoom imaging and automated analysis of endothelial cell calcium signals coincident with arteriolar dilation in Cx40(BAC) -GCaMP2 transgenic mice.

OBJECTIVE: Calcium signaling is integral to endothelium-dependent vasodilation. Our goal was to develop methods enabling automated analyses for accurately and objectively determining the dynamic relationship between EC Ca(2+) responses and arteriolar diameter in vivo. METHODS: User-friendly software (DiaFluor) written in LabView was applied to images acquired at 15fps with a custom macrozoom intravital microscope to evaluate changes in EC Ca(2+) concomitant with arteriolar diameter. Transgenic Cx40(BAC) -GCaMP2 mice expressing a fluorescent Ca(2+) indicator molecule in arteriolar ECs enabled resolution of EC Ca(2+) signaling in response to ACh microiontophoresis (500nA, 100-1000msec pulse) from a micropipette (1µm tip) positioned adjacent to an arteriole in the superfused cremaster muscle preparation. RESULTS: A 100-msec pulse of ACh (1M) had little effect on EC Ca(2+) or arteriolar diameter. As pulse duration increased, vasodilation increased with fluorescence intensity (p<0.01). Based upon fluorescence responses (F/F(o)), the effective diffusion distance of ACh along arterioles increased from ∼100µm (250msec pulse) to ∼200µm (1000msec pulse) with a peak velocity of ∼150µm/sec. CONCLUSIONS: The novel imaging and software presented here are the first to enable automated simultaneous evaluation of EC Ca(2+) signaling and endothelium-dependent vasodilation in vivo.

Evidence for impaired neurovascular transmission in a murine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle-wasting disease caused by mutations in the dystrophin gene. Little is known about how blood flow control is affected in arteriolar networks supplying dystrophic muscle. We tested the hypothesis that mdx mice, a murine model for DMD, exhibit defects in arteriolar vasomotor control. The cremaster muscle was prepared for intravital microscopy in pentobarbital sodium-anesthetized mdx and C57BL/10 control mice (n ≥ 5 per group). Spontaneous vasomotor tone increased similarly with arteriolar branch order in both mdx and C57BL/10 mice [pooled values: first order (1A), 6%; second order (2A), 56%; and third order (3A), 61%] with no difference in maximal diameters between groups measured during equilibration with topical 10 µM sodium nitroprusside (pooled values: 1A, 70 ± 3 µm; 2A, 31 ± 3 µm; and 3A, 19 ± 3 µm). Concentration-response curves to acetylcholine (ACh) and norepinephrine added to the superfusion solution did not differ between mdx and C57BL/10 mice, nor did constriction to elevated (21%) oxygen. In response to local stimulation from a micropipette, conducted vasodilation to ACh and conducted vasoconstriction to KCl were also not different between groups; however, constriction decayed with distance (P < 0.05) whereas dilation did not. Remarkably, arteriolar constriction to perivascular nerve stimulation (PNS) at 2, 4, and 8 Hz was reduced by ∼25-30% in mdx mice compared with C57BL/10 mice (P < 0.05). With intact arteriolar reactivity to agonists, attenuated constriction to perivascular nerve stimulation indicates impaired neurovascular transmission in arterioles controlling blood flow in mdx mice.