Rapid automatic assessment of microvascular density in sidestream dark field images

The purpose of this study was to develop a rapid and fully automatic method for the assessment of microvascular density and perfusion in sidestream dark field (SDF) images. We modified algorithms previously developed by our group for microvascular density assessment and introduced a new method for microvascular perfusion assessment. To validate the new algorithm for microvascular density assessment, we reanalyzed a selection of SDF video clips (n = 325) from a study in intensive care patients and compared the results to (semi-)manually found microvascular densities. The method for microvascular perfusion assessment (temporal SDF image contrast analysis, tSICA) was tested in several video simulations and in one high quality SDF video clip where the microcirculation was imaged before and during circulatory arrest in a cardiac surgery patient. We found that the new method for microvascular density assessment was very rapid (<30 s/clip) and correlated excellently with (semi-)manually measured microvascular density. The new method for microvascular perfusion assessment (tSICA) was shown to be limited by high cell densities and velocities, which severely impedes the applicability of this method in real SDF images. Hence, here we present a validated method for rapid and fully automatic assessment of microvascular density in SDF images. The new method was shown to be much faster than the conventional (semi-)manual method. Due to current SDF imaging hardware limitations, we were not able to automatically detect microvascular perfusion

Improvement of Sidestream Dark Field Imaging with an Image Acquisition Stabilizer

Background: In the present study we developed, evaluated in volunteers, and clinically validated an image acquisition stabilizer (IAS) for Sidestream Dark Field (SDF) imaging.Methods: The IAS is a stainless steel sterilizable ring which fits around the SDF probe tip. The IAS creates adhesion to the imaged tissue by application of negative pressure. The effects of the IAS on the sublingual microcirculatory flow velocities, the force required to induce pressure artifacts (PA), the time to acquire a stable image, and the duration of stable imaging were assessed in healthy volunteers. To demonstrate the clinical applicability of the SDF setup in combination with the IAS, simultaneous bilateral sublingual imaging of the microcirculation were performed during a lung recruitment maneuver (LRM) in mechanically ventilated critically ill patients. One SDF device was operated handheld; the second was fitted with the IAS and held in position by a mechanic arm. Lateral drift, number of losses of image stability and duration of stable imaging of the two methods were compared.Results: Five healthy volunteers were studied. The IAS did not affect microcirculatory flow velocities. A significantly greater force had to applied onto the tissue to induced PA with compared to without IAS (0.25 ± 0.15 N without vs. 0.62 ± 0.05 N with the IAS, p < 0.001). The IAS ensured an increased duration of a stable image sequence (8 ± 2 s without vs. 42 ± 8 s with the IAS, p < 0.001). The time required to obtain a stable image sequence was similar with and without the IAS. In eight mechanically ventilated patients undergoing a LRM the use of the IAS resulted in a significantly reduced image drifting and enabled the acquisition of significantly longer stable image sequences (24 ± 5 s without vs. 67 ± 14 s with the IAS, p = 0.006).Conclusions: The present study has validated the use of an IAS for improvement of SDF imaging by demonstrating that the IAS did not affect microcirculatory perfusion in the microscopic field of view. The IAS improved both axial and lateral SDF image stability and thereby increased the critical force required to induce pressure artifacts. The IAS ensured a significantly increased duration of maintaining a stable image sequence

Spatial Frequency-Based Analysis of Mean Red Blood Cell Speed in Single Microvessels: Investigation of Microvascular Perfusion in Rat Cerebral Cortex

BACKGROUND: Our previous study has shown that prenatal exposure to X-ray irradiation causes cerebral hypo-perfusion during the postnatal development of central nervous system (CNS). However, the source of the hypo-perfusion and its impact on the CNS development remains unclear. The present study developed an automatic analysis method to determine the mean red blood cell (RBC) speed through single microvessels imaged with two-photon microscopy in the cerebral cortex of rats prenatally exposed to X-ray irradiation (1.5 Gy). METHODOLOGY/PRINCIPAL FINDINGS: We obtained a mean RBC speed (0.9±0.6 mm/sec) that ranged from 0.2 to 4.4 mm/sec from 121 vessels in the radiation-exposed rats, which was about 40% lower than that of normal rats that were not exposed. These results were then compared with the conventional method for monitoring microvascular perfusion using the arteriovenous transit time (AVTT) determined by tracking fluorescent markers. A significant increase in the AVTT was observed in the exposed rats (1.9±0.6 sec) as compared to the age-matched non-exposed rats (1.2±0.3 sec). The results indicate that parenchyma capillary blood velocity in the exposed rats was approximately 37% lower than in non-exposed rats. CONCLUSIONS/SIGNIFICANCE: The algorithm presented is simple and robust relative to monitoring individual RBC speeds, which is superior in terms of noise tolerance and computation time. The demonstrative results show that the method developed in this study for determining the mean RBC speed in the spatial frequency domain was consistent with the conventional transit time method

Stress en toxiciteit ; de effecten van gecombineerde blootstelling aan geluid en ozon

Onderzoeksresultaten worden representeerd van de cumulatieve blootstelling aan geluid (subchronisch) en ozon (acuut) bij ratten. Geluid-blootstelling vond plaats gedurende 1 tot 3 weken bij een intensiteit van 85 tot 105 dB witte ruis middels een gerandomiseerd blootstellingsregime van 180 tot 540 minuten per dag. Blootstelling aan geluid induceerde verhogingen van de basale spiegels van corticosteron, prolactine en noradrenaline waarbij factoren als geluidsintensiteit en blootstellingsregime een belangrijke invloed hadden. Daarnaast resulteerde geluid-blootstelling in een significant veranderde reactie op een nieuwe acute stressor hetgeen aangeeft dat de fysiologische status van het dier duidelijk is veranderd. In vergelijking met blootstelling aan alleen ozon, leidde de cumulatieve blootstelling aan geluid en ozon tot significante veranderingen in de toxiciteit van ozon. Zowel met betrekking tot biochemische als immunologische variabelen, werden aanwijzingen gevonden voor een interactie tussen geluid en ozon. Echter, deze effecten waren in kwantitatief opzicht bescheiden van omvang en leidden niet tot een algemene versterking van de toxische effecten na acute ozon blootstelling. Verder onderzoek zal moeten uitwijzen wat de consequenties van de gevonden interacties zijn voor langdurig of herhaalde ozon blootstelling. In het algemeen kan worden gesteld, dat de huidige studies geen aanwijzingen hebben opgeleverd die erop duiden dat cumulatieve blootstelling aan heterogene contaminanten kan leiden tot een versterking of verandering van gezondheidseffecten.The effects of combined exposure to subchronic noise and acute ozone exposure have been investigated in rats. Noise exposure ranged from 1 to 3 weeks at intensities of 85 to 105 dB for 180 to 540 min per day in random profiles. Noise induced significant elevations in the basal levels of corticosterone, prolactin, and noradrenaline in which both intensity and the exposure profiles were important determinants. In addition, noise exposure also induced marked changes in the physiological response to a novel acute stressor indicating a changed physiological status. Compared to ozone only, the combined exposure to noise and ozone resulted in significant changes in the toxicity response. Both biochemically and immunologically indications were found for an interaction between these two factors. However, quantitatively the changes were relatively modest and the overall severity of the response to acute ozone exposure was not enhanced. Generally, the present studies did not indicate that combined exposure to heterogeneous contaminants would result in enhanced or unexpected toxicity effects and the general 'rule of assuming additivity' for combined exposure may be valid also for heterogeneous contaminants.DGM/L

Dynamic Two-Photon Imaging of Cerebral Microcirculation Using Fluorescently Labeled Red Blood Cells and Plasma

To explore the spatiotemporal dynamics of red blood cells (RBCs) and plasma flow in three-dimensional (3D) microvascular networks of the cerebral cortex, we performed two-photon microscopic imaging of the cortical microvasculature in genetically engineered rats in which the RBCs endogenously express green fluorescent protein (GFP). Water-soluble quantum dots (Qdots) were injected intravenously into the animals to label the plasma, and concurrent imaging was performed for GFP-RBCs and Qdot plasma. The RBC and plasma distributions were compared between resting state and forepaw stimulation-induced neural activation. The RBC and plasma images showed detectable signals up to a depth of 0.4 and 0.6 mm from the cortical surface, respectively. A thicker plasma layer (2-5 um) was seen in venous vessels relative to the arterial vessels. In response to neural activation, the RBCs were redistributed among the parenchymal capillary networks. In addition, individual capillaries showed a variable ratio of RBC and plasma distributions before and after activation, indicative of dynamic changes of hematocrit in single capillaries. These results demonstrate that this transgenic animal model may be useful in further investigating the mechanism that controls dynamic RBC flow in single capillaries and among multiple capillary networks of the cerebral microcirculation