Comparison of laser speckle contrast imaging with laser Doppler for assessing microvascular function

Objective

To compare the inter-day reproducibility of post-occlusive reactive hyperaemia (PORH) and sympathetic vasomotor reflexes assessed by single-point laser Doppler flowmetry (SP-LDF), integrating-probe LDF (IP-LDF) and laser speckle contrast imaging (LSCI), and the spatial variability of PORH assessed by IP-LDF and LSCI. We also evaluated the relationship between IP-LDF and LSCI perfusion values across a broad range of skin blood flows.

Methods

Eighteen healthy adults (50% male, age 27 ± 4 years) participated in this study. Using SP-LDF, IP-LDF and LSCI, indices of skin blood flow were measured on the forearm during PORH (1-, 5- and 10-min occlusions) and on the finger pad during inspiratory gasp and cold pressor tests. These tests were repeated 3-7 days later. Data were converted to cutaneous vascular conductance (CVC; laser Doppler flow/mean arterial pressure) and expressed as absolute and relative changes from pre-stimulus CVC (ΔCVC_ABS and ΔCVC_REL, respectively), as well as normalised to peak CVC for the PORH tests. Reproducibility was expressed as within-subjects coefficients of variation (CV, in %) and intraclass correlation coefficients.

Results

The reproducibility of PORH on the forearm was poorer when assessed with SP-LDF and IP-LDF compared to LSCI (e.g., CV for 5-min PORH ΔCVC_ABS = 35%, 27% and 19%, respectively), with no superior method of data expression. In contrast, the reproducibility of the inspiratory gasp and cold pressor test responses on the finger pad were better with SP-LDF and IP-LDF compared to LSCI (e.g., CV for inspiratory gasp ΔCVC_REL = 13%, 7% and 19%, respectively). The spatial variability of PORH responses was poorer with IP-LDF compared to LSCI (e.g., CV ranging 11-35% versus 3-16%, respectively). The association between simultaneous LSCI and IP-LDF perfusion values was non-linear.

Conclusion

The reproducibility of cutaneous PORH was better when assessed with LSCI compared to SP-LDF and IP-LDF; probably due to measuring larger skin areas (lower inter-site variability). However, when measuring sympathetic vasomotor reflexes on the finger pad, reproducibility was better with SP-LDF and IP-LDF, perhaps due to the high sensitivity of LSCI to changes in skin blood flow at low levels.     

Evaluation of systemic microvascular endothelial function using laser speckle contrast imaging

ObjectiveThe aim of this study was to compare cutaneous microvascular function in young healthy subjects (n = 50) with that of cardiometabolic diseased patients (n = 50) using laser speckle contrast imaging (LSCI) coupled with transdermal iontophoretic delivery of acetylcholine (ACh) and post-occlusive reactive hyperemia (PORH).MethodsCutaneous blood flow was assessed in the forearm using LSCI at rest, during PORH and during iontophoresis of ACh with increasing anodal currents of 30, 60, 90, 120, 150 and 180 μA during 10-second intervals spaced 1 min apart.ResultsEndothelium-dependent skin microvascular vasodilator responses induced by both ACh and PORH were significantly reduced in cardiometabolic diseased patients compared to healthy subjects. Vasodilator responses induced by ACh were significantly higher in young women than in young men. Iontophoresis charges up to 1.5 mC do not induce nonspecific effects on skin microvascular flux.ConclusionLSCI appears to be a promising noninvasive technique for evaluating systemic microvascular endothelial function.     

In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115(+) F4/80(+) Ly6C(+) cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases.     

Excellent reproducibility of laser speckle contrast imaging to assess skin microvascular reactivity

ObjectiveWe compared the inter-day reproducibility of post-occlusive reactive hyperemia (PORH) assessed by single-point laser Doppler flowmetry (LDF) and laser speckle contrast analysis (LSCI), and the reproducibility of local thermal hyperemia (LTH) assessed by LDF, laser Doppler imaging (LDI) and LSCI. We also tested whether skin blood flow assessment by LDF and by LSCI are correlated.MethodsSkin blood flow was evaluated during PORH and LTH using LDF, LDI (for LTH only) and LSCI on the forearms of healthy volunteers, at a 7 day interval. Data are expressed as cutaneous vascular conductance (CVC), as a function of baseline and scaled to the thermal plateau. Reproducibility is expressed as within subject coefficients of variation (CV, in %) and intra-class correlation coefficients (ICC).ResultsTwenty-eight healthy participants were enrolled in this study. The reproducibility of the PORH peak CVC was better when assessed with LSCI compared to LDF (CV = 8%; ICC = 0.76 and CV = 30%; ICC = 0.54, respectively). Inter-day reproducibility of the LTH plateau was better when assessed with LSCI or LDI than LDF (CV = 15%, ICC = 0.66; CV = 17%, ICC = 0.51 and CV = 42%, ICC = 0.28 respectively). Finally, we observed significant correlation between simultaneous LDF and LSCI measurements of the PORH peak CVC (R = 0.54; p = 0.001).ConclusionThe recently developed LSCI technique showed very good inter-day reproducibility for assessing PORH and LTH. Moreover, we showed significant correlation between LSCI and single-point LDF for PORH. However, more data are needed to evaluate the linearity between the LSCI signal and skin blood flow.     

Skin microdialysis coupled with laser speckle contrast imaging to assess microvascular reactivity

Objective

Laser Speckle Contrast Imaging (LSCI) can be used to assess real-time responses of skin microcirculation to pharmacological interventions. The main objective of this study was to determine whether intradermal or subdermal microdialysis fiber insertion, coupled with skin flux recording using LSCI, can be used to assess baseline cutaneous flux and the post-occlusive reactive hyperemic response. The microdialysis sites were compared to control area without microdialysis fibers.

Methods

One dermal and two subdermal microdialysis fibers were randomly inserted in the right forearm skin of six healthy volunteers. We performed consecutively tests of post-occlusive hyperemia, infusion of 29 mM sodium nitroprusside (SNP), local thermal hyperemia at 43 °C and a second 29 mM SNP infusion at the end of the experiment.

Results

Two hours after fiber insertion, cutaneous vascular conductances (CVC) at the subdermal fiber sites were not different from their respective control regions of interest, while at the dermal site CVC remained higher (0.48 +/− 0.15 versus 0.37 +/− 0.1 PU.mm Hg⁻¹, P = 0.003). The peak CVC and area under the curve observed during post-occlusive reactive hyperemia were similar at all fiber sites and their respective controls. We observed a similar increase in CVC using 29 mM SNP infusion, 40 min local heating at 43 °C, and their combination. Finally, physiological and pharmacological responses of the subdermal sites were reproducible in terms of amplitude, whether expressed as raw CVC or as %CVCmax.

Conclusions

We showed that studying skin microvascular physiological or pharmacological responses using inserted subdermal microdialysis fibers coupled with LSCI is feasible and reproducible, and provides two-dimensional information. This technique will be useful for future mechanistic studies of skin microcirculation.     

A reduced gastric corpus microvascular blood flow during Ivor-Lewis esophagectomy detected by laser speckle contrast imaging technique

Background: Reduced microvascular blood flow is related to anastomotic insufficiency following esophagectomy, emphasizing a need for intraoperative monitoring of the microcirculation. This study evaluated if laser speckle contrast imaging (LSCI) was able to detect intraoperative changes in gastric microcirculation.

Methods: Gastric microcirculation was assessed prior to and after reconstruction of gastric continuity in 25 consecutive patients operated for adenocarcinoma with open Ivor-Lewis esophagectomy while hemodynamic variables were recorded.

Results: During upper laparotomy, microcirculation at the corpus decreased by 25% from baseline to mobilization of the stomach (p?=?.008) and decreased further (to a total decrease of 40%) following gastric pull to the thorax (p?=?.013). On the other hand, microcirculation at the antrum did not change significantly after gastric mobilization (p?=?.091). The decrease in corpus microcirculation took place unrelated to central cardiovascular variables.

Conclusion: Using LSCI technique, we identified a reduced microcirculation at the corpus area during open Ivor-Lewis esophagectomy. LSCI provides an option for real-time assessment of gastric microcirculation and could form basis for intraoperative stabilization of the microcirculation.     

Intussusceptive angiogenesis: expansion and remodeling of microvascular networks

Intussusceptive angiogenesis is a dynamic intravascular process capable of dramatically modifying the structure of the microcirculation. The distinctive structural feature of intussusceptive angiogenesis is the intussusceptive pillar—a cylindrical microstructure that spans the lumen of small vessels and capillaries. The extension of the intussusceptive pillar appears to be a mechanism for pruning redundant or inefficient vessels, modifying the branch angle of bifurcating vessels and duplicating existing vessels. Despite the biological importance and therapeutic potential, intussusceptive angiogenesis remains a mystery, in part, because it is an intravascular process that is unseen by conventional light microscopy. Here, we review several fundamental questions in the context of our current understanding of both intussusceptive and sprouting angiogenesis. (1) What are the physiologic signals that trigger pillar formation? (2) What endothelial and blood flow conditions specify pillar location? (3) How do pillars respond to the mechanical influence of blood flow? (4) What biological influences contribute to pillar extension? The answers to these questions are likely to provide important insights into the structure and function of microvascular networks.     

‘In vivo’ optical approaches to angiogenesis imaging

In recent years, molecular imaging gained significant importance in biomedical research. Optical imaging developed into a modality which enables the visualization and quantification of all kinds of cellular processes and cancerous cell growth in small animals. Novel gene reporter mice and cell lines and the development of targeted and cleavable fluorescent “smart” probes form a powerful imaging toolbox. The development of systems collecting tomographic bioluminescence and fluorescence data enabled even more spatial accuracy and more quantitative measurements. Here we describe various bioluminescent and fluorescent gene reporter models and probes that can be used to specifically image and quantify neovascularization or the angiogenic process itself.     

Akt1 is necessary for the vascular maturation and angiogenesis during cutaneous wound healing

Previous in vivo and in vitro studies have shown that Akt1 serves as a crucial regulator of vascular maturation, extracellular matrix composition, and angiogenesis in tumors. Hence, we hypothesized that Akt1 may be necessary for other angiogenesis-dependent processes, including wound healing. Using Akt1 (-/-) and Akt2 (-/-) mice, we demonstrate that deficiency of Akt1, but not Akt2, results in impaired assembly of collagen in skin wounds and around the blood vessels. Wounds in Akt1 (-/-) mice, but not in Akt2 (-/-) mice, were characterized by reduced vascular area as well as impaired vascular maturation as evidenced by reduced smooth muscle cell recruitment. Expression level of a major angiogenic growth factor, VEGF, was significantly lower in wound tissues of Akt1 (-/-) mice as compared to WT. However, despite the impaired collagen assembly and reduced angiogenesis in Akt1 (-/-) wounds, no significant difference in migration of fibroblasts into the wound area was observed between WT and Akt1 (-/-) mice. Importantly, the dynamics of wound closure were similar between WT, Akt1 (-/-), and Akt2 (-/-) mice. Thus, it appears that although the lack of Akt1 impairs VEGF expression, wound angiogenesis, and subsequent maturation of vasculature, it has no effect on the wound closure. These findings may have clinical applications for the improvement of treatment procedures with reported history of wound healing complications.     

Comparison of infrared thermography and laser speckle contrast imaging for the dynamic assessment of digital microvascular function

ObjectivesLaser speckle contrast imaging (LSCI) is a novel non-invasive microvascular imaging modality. The present study evaluates the validity and reliability of LSCI by comparison with infrared thermography (IRT) for the dynamic assessment of digital microvascular function in healthy volunteers.MethodsSubjects attended on 3 occasions. Simultaneous assessment of cutaneous perfusion at 3 distinct regions of interest (ROI) within the hands was undertaken using LSCI and infrared thermography (IRT) at baseline, and at 13 s intervals over 15 min following a standardised local cold challenge. Endpoints for evaluation included absolute measurements at baseline and following cold stress, in addition to the characteristics of the re-warming curves (maximum % recovery and maximum gradient). Visits 1 and 2 were undertaken in identical conditions (ambient temperature 23 °C) to assess reproducibility, whereas visit 3 was undertaken at a lower ambient room temperature of 18 °C to evaluate responsiveness to reduction in ambient room temperature.ResultsFourteen healthy participants completed the study. There was greater variability in the data generated using LSCI compared with the highly damped IRT, reflecting greater sensitivity of LSCI to physiological variation and movement artefact. LSCI and IRT correlated well at baseline and following cold challenge for all endpoints (r_s for pooled data between 0.5 and 0.65, p < 0.00005). Reproducibility of both IRT and LSCI was excellent (ICCs > 0.75) for absolute assessments but lower for re-warming curve characteristics. LSCI provides greater spatial resolution than IRT identifying variation in cutaneous perfusion within the hands most likely associated with the presence of arteriovenous anastamoses. Both techniques were responsive to reduction in ambient room temperature. Effect sizes were greatest for IRT than LSCI (e.g. ? 1.17 vs. ? 0.85 at ROI 1 at baseline) although this may represent heat transfer rather than altered vascular perfusion.DiscussionIn the dynamic assessment of digital vascular perfusion, LSCI correlates well with IRT, is reproducible and responsive to reduction in ambient room temperature. Absolute measurements appear preferable to parameters derived from re-warming curve characteristics when assessing digital perfusion following cold challenge. The greater temporal and spatial resolution of LSCI compared with IRT may facilitate the development of novel assessment tools of autonomic function and digital cutaneous perfusion.     

In vivo two-photon voltage-sensitive dye imaging reveals top-down control of cortical layers 1 and 2 during wakefulness

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly along the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatiotemporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 mum) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4- to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5- to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.     

Acute hemodynamic changes during intravenous dipyridamole thallium imaging early after infarction

In order to determine the safety and hemodynamic effects of intravenous dipyridamole infusion for thallium-201 scinitigraphy in patients with acute ischemic syndromes, 10 patients with recent uncomplicated myocardial infarction (7 +/- 2 days pre-test) had central pressures and cardiac output values measured serially in a coronary care unit during and after the administration of dipyridamole (0.56 mg/kg over 4 minutes) and following aminophylline reversal (50 to 150 mg intravenously) of dipyridamole effect. Cardiac medications were not discontinued. Double product did not change significantly (8522 +/- 1811 versus 9044 +/- 1701; p = NS). Serious ischemic events did not occur, although 20% of patients had noncardiac side effects and 30% developed greater than or equal to 1 mm ST segment depression with associated angina in one-third of these cases. The peripheral blood pressure and heart rate response did not predict the occurrence of myocardial ischemia. Dipyridamole significantly reduced systemic vascular resistance (1218 +/- 302 to 739 +/- 166 dyne/sec-1/cm-5; p less than 0.05) and increased cardiac index (3.1 +/- 0.7 to 4.7 +/- 1.0 L/min/m2; p less than 0.05) within approximately 10 minutes, in association with a significant increase in pulmonary capillary wedge pressure (13 +/- 5 to 17 +/- 6 mm Hg; p less than 0.05). Three patients developed silent new "V" waves in their pulmonary capillary wedge pressure tracing, associated with anterior thallium redistribution. All three patients with newly elevated wedge pressures (greater than 15 mm Hg) had both thallium-201 redistribution and multivessel coronary disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo detection of morphological and microvascular changes of the colon in association with colitis using fiberoptic confocal imaging (FOCI)

Using a well-established rodent model of inflammatory bowel disease (IBD), the present study examined changes in the microvasculature of the colonic mucosa in association with ulcerative colitis (UC). The results were compared to microscopic alterations in tissue morphology to establish a temporal relationship between microcirculatory dysfunction and IBD pathology. Mild colitis was induced in rats by the oral consumption of 5% dextran sulfate sodium (DSS) in drinking water. Control animals were provided with water ad libitum. After 3, 5, and 7 days of oral ingestion of DSS, anesthetized rats were laparotomized. The mucosal surface of the distal colon was then examined using fiber optic confocal imaging (FOCI; excitation 488 nm argon ion laser, detection above 515 nm). Changes in the mucosal architecture were examined following the topical application of the fluorescent dye, tetracycline hydrochloride. Tetracycline hydrochloride, an antibiotic used widely in clinical medicine, enabled imaging of the crypts at the surface of the mucosa. Spatial changes in the microvascular structure were assessed following the intravenous administration of fluorescein isothiocyanate dextran (FITC-dextran). Confocal images were correlated with clinical parameters, including weight loss, occult blood, and stool consistency. Attenuation of the colonic epithelium was detected on day 3 colitis. Morphological changes including crypt loss, crypt distortion, and inflammatory cell infiltrate were detected on day 5 and day 7 colitis. Dual channel imaging showed the mucosal capillary network outlining the stromal confines of the mucus-secreting glands in control tissue. Experimental colitis resulted in diffuse hypervascularity and tortuosity of the capillary vessels. Evidence of increased vessel leakiness (leakage of FITC-dextran from the lumen) was first detected on day 5 colitis. Complete disruption of the normal honeycomb pattern of the vessels and capillary dilation was evident after 7 days of DSS ingestion. These findings suggest that the pathogenesis of ulcerative colitis is associated with changes in the vascular architecture as demonstrated in vivo using confocal microscopy.     

In vivo imaging of the molecular distribution of the VEGF receptor during angiogenesis in a mouse model of ischemia

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis and has been applied to medical therapy. However, because vascular imaging at the molecular level is impossible, the detailed in vivo dynamics of VEGF and its receptor (VEGFR) remain unknown. In this study, to understand the molecular distribution of VEGF and the VEGFR, we prepared ischemic mice with a new surgical method and induced angiogenesis in the gastrocnemius muscle. Then, we made a VEGF-conjugated fluorescence nanoparticle and performed staining of VEGFR-expressing cells with the fluorescent probe, demonstrating the high affinity of the probe for VEGFR. To observe the physiologic molecular distribution of VEGFR, we performed in vivo single-particle imaging of gastrocnemius in the ischemic leg with the fluorescent probe. The results suggested that only a 3-fold difference of VEGFR distribution is involved in the formation of branched vasculature in angiogenesis, although previous ex vivo data showed a 13-fold difference in its distribution, indicating that a method inducing a several-fold local increase of VEGFR concentration may be effective in generating site-specific angiogenesis in ischemic disease. This new in vivo imaging of ischemic mice could make useful contributions to understanding the mechanisms of angiogenesis and to developing a VEGFR-related drug.