Body mapping of human cutaneous microcirculatory perfusion using a real-time laser Doppler imager.

There are many pathologies and/or surgical situations where understanding how cutaneous capillary blood flow is behaving would be of clinical benefit. Laser Doppler imaging (LDI) has long been considered an ideal candidate for this yet was never widely adopted. The technology was deemed too slow, needing minutes to record one image. Here, we present the use of a new, real-time LDI, which is sensitive enough to resolve pulsatile blood flow in the microcirculation and can record images and videos instantaneously. We show that when comparing the ratios of absolute perfusion images from different regions of the body, remarkably reproducible data can be obtained when looking at a control population of male volunteers. In the future, we plan to add more control and diseased groups, effectively giving the clinician a non-invasive, safe and easy-to-use diagnostic for assessing the extent of microcirculatory disorders and high-risk surgical situations.

Early Venous Occlusion Detection in a Free Flap Using Real-time Laser Doppler Imaging.

SUMMARY: Early detection of venous occlusion in free flaps is particularly difficult to identify, and its duration is known to be directly proportional to flap mortality. Here, we report a case of deep inferior epigastric perforator based breast reconstruction in which the intraoperative use of a perfusion camera enabled identifying a venous occlusion based on microcirculatory pulsation dynamics in real time. The sensitivity of our proposed method suggests that in certain cases in which the onset of venous occlusion begins in the operating room we can detect and treat occlusion before sending the patient to recovery. Further development of this technique will allow for earlier and more objective decision making with regard to venous occlusion detection in free tissue transfer.

Real-time full field laser Doppler imaging.

We present a full field laser Doppler imaging instrument, which enables real-time in vivo assessment of blood flow in dermal tissue and skin. This instrument monitors the blood perfusion in an area of about 50 cm(2) with 480 × 480 pixels per frame at a rate of 12-14 frames per second. Smaller frames can be monitored at much higher frame rates. We recorded the microcirculation in healthy skin before, during and after arterial occlusion. In initial clinical case studies, we imaged the microcirculation in burned skin and monitored the recovery of blood flow in a skin flap during reconstructive surgery indicating the high potential of LDI for clinical applications. Small animal imaging in mouse ears clearly revealed the network of blood vessels and the corresponding blood perfusion.

Early detection of microcirculatory perfusion changes with a high resolution, real time laser Doppler imaging camera--frostbite case study.

A 41-year-old male presented with severe frostbite that was monitored clinically and with a new laser Doppler imaging (LDI) camera that records arbitrary microcirculatory perfusion units (1-256 arbitrary perfusion units (APU's)). LDI monitoring detected perfusion differences in hand and foot not seen visually. On day 4-5 after injury, LDI showed that while fingers did not experience any significant perfusion change (average of 31±25 APUs on day 5), the patient's left big toe did (from 17±29 APUs day 4 to 103±55 APUs day 5). These changes in regional perfusion were not detectable by visual examination. On day 53 postinjury, all fingers with reduced perfusion by LDI were amputated, while the toe could be salvaged. This case clearly demonstrates that insufficient microcirculatory perfusion can be identified using LDI in ways which visual examination alone does not permit, allowing prognosis of clinical outcomes. Such information may also be used to develop improved treatment approaches.

Regulation of arginase pathway in response to wall shear stress.

OBJECTIVE: Alterations of wall shear stress can predispose the endothelium to the development of atherosclerotic plaques. Ample evidence indicates that arginase expression and/or activity correlates with several risk factors for cardiovascular disease including atherosclerosis. We investigated the regulation of arginase pathway in response to distinct patterns of wall shear stress. METHODS: Isolated porcine endothelial cells and carotid arterial segments were perfused under unidirectional high shear stress (HSS) or oscillatory shear stress (OSS) for 1 and 3 days. Arginase I and II expression, cellular localization and enzyme activity were, respectively, assessed by Western blot, immunohistochemistry and colorimetric determination of urea. The contribution of arginase to the processes of endothelial dysfunction, cell proliferation and arterial remodeling induced by OSS was evaluated by administration of the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-Noha). RESULTS: Only arginase II isoform was detected on porcine carotid endothelial cells and on carotid artery. Exposure of arteries to OSS increased arginase II expression and activity as compared to HSS. Inhibition of arginase by nor-Noha improved NO-dependent endothelial function and decreased total vascular ROS formation in arteries submitted to OSS. In addition, inhibition of arginase activity decreased smooth muscle cell proliferation rate with no effect on collagen content after OSS. CONCLUSIONS: Exposure of carotid artery to oscillatory flow induced a more pronounced activation of arginase as compared to HSS. Inhibition of arginase in arteries exposed to OSS improved NO-dependent endothelial function and decrease smooth muscle cell proliferation rate, both processes are important for the focal development of atherosclerotic plaque.

Reduced cyclic stretch, endothelial dysfunction, and oxidative stress: an ex vivo model.

BACKGROUND: The objective of this study was to investigate whether reduction of cyclic circumferential stretch will impair endothelial function and elevate basal levels of oxidative stress, both known risk factors linked to cardiovascular disease. METHODS: Ex vivo and in vitro models were used to perfuse porcine carotid arteries and porcine endothelial cells, respectively, for 24 h. In both cases, one group was allowed to stretch naturally when exposed to a pulse shear stress (6+/-3 dynes/cm(2)) combined with a pulse pressure of 80+/-10 mmHg, yielding a physiological cyclic stretch of 4-5%. This group was compared to a reduced stretch group, achieved by wrapping the arterial segment with a silicon band or by seeding the endothelial cells inside less compliant tubes, decreasing cyclic stretch to 1%. RESULTS: The experimentally reduced compliance caused a significant decrease in bradykinin-dependent vascular relaxation. Reduced compliance significantly decreased the phosphorylation of serine 1177 (Ser1177) on eNOS, suggesting the activity of eNOS was decreased. Overall production of reactive oxygen species was increased by reducing compliance, as visualized with DHE. Finally, p22-phox and p47-phox, key players in the superoxide-generating NAD(P)H oxidase, were also up-regulated by reduced compliance. CONCLUSIONS: These findings point out how reduced arterial compliance increases the risk of arterial disease by creating a less functional endothelium, interrupting the eNOS activation pathway, and increasing the vascular levels of oxidative stress.

Autonomous effects of shear stress and cyclic circumferential stretch regarding endothelial dysfunction and oxidative stress: an ex vivo arterial model.

Cyclic circumferential stretch and shear stress caused by pulsatile blood flow work in concert, yet are very different stimuli capable of independently mediating endothelial function by modulating eNOS expression, oxidative stress (via production of superoxide anion) and NO bioavailability. Porcine carotid arteries were perfused using an ex vivo arterial support system for 72 h. Groups we created by combining normal (5%) and reduced (1%) stretch with high shear (6 +/- 3 dynes/cm(2)) and oscillatory shear (0.3 +/- 3 dynes/cm(2)) stress while maintaining a pulse pressure of 80 +/- 10 mm Hg. Oscillatory flow and reduced stretch both proved detrimental to endothelial function, whereas oscillatory flow alone dominated total endogenous vascular wall superoxide anion production. Yet, when superoxide anion production was analyzed in just the endothelial region, we observed that it was modulated more significantly by reduced cyclic stretch than by oscillatory shear, emphasizing an important distinction between shear- and stretch-mediated effects to the vascular wall. Western blotting analysis of eNOS and nitrotyrosine proved that they too are more significantly negatively modulated by oscillatory flow than by reduced stretch. These findings point out how shear and stretch stimulate regions of the vascular wall differently, affecting NO bioavailability and contributing to vascular disease.

Differential effects of reduced cyclic stretch and perturbed shear stress within the arterial wall and on smooth muscle function.

BACKGROUND: Cyclic circumferential stretch and shear stress act in concert and yet are capable of independently mediating arterial smooth muscle function, modulating the production of superoxide and stimulating arterial remodeling. METHODS: Porcine carotid arteries were perfused ex vivo for 72 h. Groups combining normal (5%) and reduced (1%) stretch with high shear (6 +/- 3 dyn/cm2) and oscillatory shear (0.3 +/- 3 dyn/cm2) stress were created, while maintaining a pulse pressure of 80 +/- 10 mm Hg. RESULTS: Total superoxide production, fibronectin expression, and gelatinase activation were mediated by shear stress, but expression in the endothelial region was mediated by reduced cyclic stretch. By plotting intensity vs. radius, we saw that superoxide and gelatinase activity were in part mediated by stress distributions throughout the vascular wall, whereas fibronectin and p22-phox were much less or not at all. These findings, when coupled with our results from tissue reactive studies, suggest that the arterial remodeling process triggered in the endothelial region due to reduced stretch causes the most significant changes in arterial smooth muscle function. CONCLUSIONS: We have found that the remodeling process triggered by reduced compliance in the endothelial region of large conduit arteries has a more profound detrimental effect to smooth muscle function than that brought on by perturbed shear stress. This work provides new insight by suggesting that although mechanical stimuli such as cyclic stretch and shear stress are known to augment similar markers of vascular remodeling, the location of their expression throughout the vascular wall differs greatly and this can have dramatic effects on vascular function.

Micelles for delivery of nitric oxide.

We designed block copolymer pro-amphiphiles and amphiphiles for providing very long-term release of nitric oxide (NO). A block copolymer of N-acryloylmorpholine (AM, as a hydrophile) and N-acryloyl-2,5-dimethylpiperazine (AZd, as a hydrophilic precursor) was synthesized. The poly(N-acryloyl-2,5-dimethylpiperazine) (PAZd) is water-soluble, but chemical reaction of the secondary amines with NO to form a N-diazeniumdiolate (NONOate) converts the hydrophilic PAZd into a hydrophobic poly(sodium-1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-1,2-diolate) (PAZd.NONOate), driving aggregation. The PAM block guides this process toward micellization, rather than precipitation, yielding ca. 50 nm spherical micelles. The hydrophobic core of the micelle shielded the NONOate from the presence of water, and thus protons, which are required for NO liberation, delaying release to a remarkable 7 d half-life. Release of the NO returned the original soluble polymer. The very small NO-loaded micelles were able to penetrate complex tissue structures, such as the arterial media, opening up a number of tissue targets to NO-based therapy.

Effects of reduced cyclic stretch on vascular smooth muscle cell function of pig carotids perfused ex vivo.

BACKGROUND: With advancing age arteries stiffen, reducing arterial compliance and leading to the development of systolic hypertension and to a substantial increase in pulse pressure. An augmented pulse pressure can be a predictor of the development of hypertension, which has been linked to several cardiovascular diseases including atherosclerosis, and to pathologies such as diabetes and renal dysfunction. In this study, we tested the hypothesis that reduced wall compliance induces pulse-pressure-mediated changes in arterial wall metabolism and remodeling. METHODS: Porcine carotid arteries were perfused for 24 h using an ex vivo arterial support system. Control arteries were exposed to a pulse shear stress (6 +/- 3 dynes/cm(2)) combined with a pulse pressure of 80 +/- 10 mm Hg, yielding a physiological cyclic stretch of 4-5%. A reduced compliance group was also studied, in which arteries were wrapped with an external band, thereby decreasing cyclic stretch to levels <1%. RESULTS: The experimentally reduced compliance caused a decreased contraction capacity induced by norepinephrine(NE), and this was associated with lower levels of alpha-smooth muscle cell-actin (alpha-SMC-actin) and desmin protein expressions. Arteries that were exposed to a reduced cyclic stretch exhibited a higher level of matrix metalloproteinase-2 (MMP-2) expression activity as well as an increase in Ki67 expression, thereby suggesting that matrix degradation and cellular proliferation had been initiated. Furthermore, the expression of plasminogen activator inhibitor-1 (PAI-1) in stiffened arteries was lower than in the control arteries. CONCLUSIONS: These findings underline the importance of cyclic stretch in the maintenance of a differentiated and fully functional phenotype of vascular SMCs, as well as in the regulation of migratory properties, proliferation, and matrix turnover.