Video Microscopy System Research Articles

A Morphometric Study of the Pars Plana of the Ciliary Body in Human Cadaver Eyes.

This study aimed to determine the pars plana length in postmortem human eyes using advanced morphometric techniques and correlate demographics to ocular metrics such as age, sex, ethnicity, and axial length. Between February and July 2005, we conducted a cross-sectional observational study on 46 human cadaver eyes deemed unsuitable for transplant by the SBO Eye Bank. The morphometric analysis was performed on projected images using a surgical microscope and a video-microscopy system with a 20.5:1 correction factor. The pars plana length was measured three times per quadrant, with the final value being the mean of these measurements. Of the 46 eyes collected, 9 were unsuitable for the study due to technical constraints in conducting intraocular measurements. Overall, the average axial length was 25.20 mm. The average pars plana length was 3.8 mm in all quadrants, with no measurements below 2.8 mm or above 4.9 mm. There were no statistically significant variations across quadrants or with age, sex, axial length, or laterality. Accurately defining the pars plana dimensions is crucial for safely accessing the posterior segment of the eye and minimizing complications during intraocular procedures, such as intravitreal injections and vitreoretinal surgeries.

The effect of triterpene glycoside on survival and changes in the cytokine profile in the spleen of BALB/c mice under conditions of lethal influenza infection A/WSN/1/33(H1N1)

BACKGROUND: Currently, the search and development of compounds, including herbal ones, with anti-influenza activity are relevant.
 AIM: To study the effect of oral administration of the triterpene glycoside saponin tauroside Sx1 on survival and changes in the cytokine profile of the spleen of mice under conditions of lethal influenza infection.
 MATERIAL AND METHODS: 78 male BALB/c mice aged 4–6 weeks, weighing 16–18 g, were used. They were divided into groups: first — control group; the second was a group of animals infected with influenza virus A/WSN/1/33(H1N1); the third was a group of infected animals that received saponin tauroside Sx1, isolated from the Crimean ivy Hedera taurica (Hibberd) Carrière at a dose of 11.8 mg/kg per day against the background of a viral infection for 3 days. The survival and expression of interleukins-1β and -10 in the spleen of mice on the 4th and 14th days of the experiment were studied. Spleen fragments were stained with hematoxylin and eosin. Microscopic analysis was performed using a video microscopy system. Immunohistochemical study was performed according to a standard protocol. Intergroup differences were assessed using the Kruskal–Wallis test and the Mann–Whitney U test using MS Office (Excel 2010) and Statistica 10.0.
 RESULTS: Administration of saponin tauroside Sx1 significantly increased the average life expectancy of animals by 4.6 days compared to the group without correction; survival rate was 30.0% (p=0.0233). Against the background of influenza infection, an increase in the number of cells secreting interleukin-1β was detected in the spleen of mice. In the mantle zone of the second group, the level of cytokine expression exceeded the control values by 3.41 times (p=0.0001), and the values of the third group — by 3.38 times (p=0.0001). By the 14th day of the experiment in the mantle zone, expression in the group with saponin administration increased, exceeding the indicators of the group without correction by 34.78% (p=0.0482). By the 4th day of the experiment, in both experimental groups, the number of cells positive for interleukin-10 increased in the mantle zone and decreased compensatoryly in the marginal zone. By the 14th day, in the group without correction, a decrease in expression by 24.86% (p=0.0487) was recorded; after the administration of saponin, the number of immunopositive cells was practically no different from the control (p=0.0443).
 CONCLUSION: Therapeutic administration of saponin tauroside Sx1 stabilizes the cytokine profile of the spleen of mice and promotes their better survival in conditions of lethal influenza infection.

Interobserver reliability for manual analysis of sidestream darkfield videomicroscopy clips after resizing in ImageJ

BackgroundDirect assessment of microcirculatory function remains a critical care research tool but approaches for analysis of microcirculatory videomicroscopy clips are shifting from manual to automated algorithms, with a view to clinical application in the intensive care unit. Automated analysis software associated with current sidestream darkfield videomicroscopy systems is demonstrably unreliable; therefore, semi-automated analysis of captured clips should be undertaken in older generations of software. We present a method for capture of microcirculatory clips using current version videomicroscope hardware and resizing of clips to allow compatibility with legacy analysis software. The interobserver reliability of this novel approach is examined, in addition to a comparison of this approach with the current generation of automated analysis software.ResultsResizing microcirculatory clips did not significantly change image quality. Assessment of bias between observers for manual analysis of resized clips; and between manually analysed clips and automated software analysis was undertaken by Bland–Altman analysis. Bias was demonstrated for all parameters for manual analysis of resized clips (total vessel density = 6.8, perfused vessel density = 6.3, proportion of perfused vessels = − 8.79, microvascular flow index = − 0.08). Marked bias between manual analysis and automated analysis was also evident (total vessel density = 16.6, perfused vessel density = 16.0, proportion of perfused vessels = 1.8). The difference between manual and automated analysis was linearly related to the magnitude of the measured parameter.ConclusionsPoor reliability of automated analysis is a significant hurdle for clinical translation of microcirculatory monitoring. The method presented here allows capture of microcirculatory clips using current hardware that are backwards compatible with older versions of manual analysis software. We conclude that this approach is appropriate for research applications in the intensive care unit, however the time delay to results limits utility for clinical translation.

Predictors of Requirement of Inotrope Among Patients With Early Sepsis: Special Reference to Microcirculatory Parameters

IntroductionThe management of septic shock and refractory septic shock is essential in preventing sepsis-related death. The handheld vital microscope is a new modality of investigation for sepsis for microcirculatory assessment. This study aimed to identify predictors of inotrope requirements among patients with early sepsis and impending septic shock with particular reference to sublingual microcirculation assessment parameters.MethodologyWe conducted an observational cross-sectional hospital-based study in central India. The formal sample size was calculated to be 52 patients using a convenient sampling technique. The study was initiated with ethics approval (IHEC-LOP/2019/ MD0090) with consent from the patients. We used the MicroScan (MicroVision Medical, Netherlands) Video Microscope System (No.16A00102) to obtain sidestream dark-field imaging along with the AVA 4.3C software (MicroVision Medical).ResultsOf 51 cases, 60.8% were women, and 39.2% were men, and the study population had a mean age of 41.0 ± 14.9 years. Patients were recruited from medical wards (64.7%) and emergency departments (35.3%). The most common site of infection was gastrointestinal (33.3%), followed by respiratory infections (25.5%) and genitourinary infections (11.8%). The quick sequential organ failure assessment score was 2.0 ± 0.1. Eight patients required inotropes, and six patients died. High respiratory rates and lactate levels were important predictors of inotrope requirements in patients with early sepsis. Sublingual microcirculatory parameters at baseline did not significantly affect the requirement of inotropes consequently.ConclusionsSublingual microscopy is a suggested tool for the management of sepsis. However, without clearly defined cut-off values, handheld vital microscopy could not predict fluid responsiveness among patients with early sepsis. Also, it would be difficult to incorporate this technology into regular practice without equipment upgrades and image acquisition software.

Investigation of the relationship between corpse age and microscopic morphometric indices of the fourth and sixth rib bones

ABSTRACT This study was performed to estimate the corpse age using histological examinations of the fourth and sixth ribs by a morphometric method. One hundred samples of referred bodies of the Legal Medicine Organization of Lorestan province, Iran, were examined. The sampling was performed from the fourth and sixth ribs. The transverse sections of the samples were prepared, in which various variables such as the diameter of the haversian system (DHC), haversian system perimeter (PHS), haversian system area (AHS), and the thickness of the haversian lamellae (TH) were measured using a Motic microscope video system and the Motic Image Plus 2 software. To predict age from sex and the morphometric parameters a stepwise multiple linear regression was used. Accordingly, AHS in rib 4, DHC with lens 10 in rib 4, DHC with lens 10 in rib 6 and TH in rib 6 remained in the model at significance level 0.05 (R square = 0.1654). Although age was associated with some of the studied parameters, the obtained equation was not fit enough to predict the exact age. Further parameters should be studied for prediction of age in corpse.

CS-5 A case of anaplastic pleomorphic xanthoastrocytoma in the pineal region

Tumors in the pineal gland are rare tumors that account for about 0.3% of all brain tumors and have various histological types of tumors develop with germinoma, pineocytoma, and pineoblastoma in that order. On the other hand, pleomorphic xanthoastrocytoma (PXA) is a rare tumor of less than 0.2% and frequently occurs in supratentorial cerebral surface of children and young adults.A case was a 61-year-old man whose pineal tumor was found due to visual disturbance. MRI showed a 23 mm-sized lesion with cysts and inhomogeneous enhancement in the pineal gland. Partial calcification was observed, but there was no non-communicating hydrocephalus, and no increase in HCG-β and AFP with blood sampling. A midline suboccipital craniotomy was performed in the sitting position, a head-up surgery was performed using a 4K / 3D video microscope system (ORBEYE exoscope, Olympus) by the infratentorial supracerebellar approach (ITSCA). The bridging veins and precentral cerebellar vein were dissected to expose the posterior surface of the tumor, and internal decompression was performed. For the complication of air embolism, artificial cerebrospinal fluid was sprayed, and the bilateral internal jugular veins were compressed to confirm the inflow point. There was strong adhesion to the Rt vein of Rosenthal, and the site was removed intracapsularly. Finally, subtotal resection was performed with remaining the upper part of the tumor, a blind lesion behind the Vein of Galen. Vertical gaze palsy occurred after this operation, but it gradually improved over time. Tumor showed pathologically remarkable polymorphism, poor microvascular proliferation and necrosis, but mitotic figures 4–5 / 10HPF, MIB-1 index 10%, GFAP positive, no BRAF V600E mutation. There are few reports of PXA occurring in the pineal gland, and this case is the sixth case. It is also the first report for pineal tumors using ORBEYE through ITSCA in the sitting position.

Formulation characteristics of monodisperse structured lipid microparticles using microchannel emulsification

Structured lipid microparticles (StLMs) composed of oil and solid lipid in specific ratios are drawing significant attention, owing to their stability in loading and controllability in releasing bioactive compounds. In this study, we formulated monodisperse StLMs with palmitic acid or tripalmitin mixed with soybean oil in different ratios using microchannel emulsification (MCE) at ∼70 °C and following crystallization by cooling. Based on the physicochemical properties of the different dispersed phases, the formulation characteristics were observed and investigated using a microscope video system connected to the MCE system. For each ratio and composition at ∼70 °C, the lipid droplets presented average diameter and coefficient of variation (CV) values in respective ranges of 15.5–19.0 μm and 3.3–8.4%. Following droplet crystallization by cooling, the StLMs exhibited morphological changes but negligible changes in average size and CV. From the internal structure of each lipid molecular and the fundamental information supplied by differential scanning calorimetry (DSC) experiments, we outline the assumptions for morphology variation, which can be used as reference for encapsulation research of StLMs.

Confocal Blood Flow Videomicroscopy of Thrombus Formation over Human Arteries and Local Targeting of P2X7.

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

IMPROVEMENT OF METHOD FOR PRODUCING CHEMICAL ABSORBER OF AMMONIA AND HYDROGEN SULFIDE

The method of obtaining a chemical adsorbent of ammonia and hydrogen sulfide is proposed to be modified to improve sorption characteristics. The dependence of the kinetics of the process was studied - crystallization of solutions of copper (II) sulfate with a concentration of 2.19 mol/dm3 and 2.38 mol/dm3 depending on the method of preparation of the impregnating solution: thermal with heating to a temperature of 80 - 90 °C, using ultrasonic oscillations with a frequency of 22 kHz and an intensity of 3.5 W/cm2, and the dependence of the studied thermal method with the additional introduction of 0.01% surfactants into the solution with nonionic, anionic and cationogenic properties. Crystallization of copper sulfate was performed using a probe video microscopy system. The formation of smaller crystals is observed upon cooling of the solutions subjected to ultrasound. The introduction of surfactants did not affect the size of the crystals of copper (II) sulfate. The dependence of the physicochemical properties of a solution of copper (II) sulfate at a concentration of 1.17 mol/dm3 and 2.26 mol/dm3 on the introduction of 0.01% surfactant and a change in the temperature of the solution was studied. It was found that the introduction of a surfactant can effectively reduce the surface tension of the solution at a temperature of 80 °C and reduce the contact angle of the solution of activated carbon granules. Impregnating solutions of copper (II) sulfate are prepared using the above methods and samples of a chemical adsorbent of ammonia and hydrogen sulfide are made by impregnating granular activated carbon with the obtained solutions. It is proved that the use of surfactants and ultrasonic vibrations during the preparation of an impregnating solution allows to increase the amount of active form of copper on the surface of the carrier during impregnation, which helps to increase the dynamic capacity of chemisorbents.

Manual and Automatic Image Analysis Segmentation Methods for Blood Flow Studies in Microchannels.

In blood flow studies, image analysis plays an extremely important role to examine raw data obtained by high-speed video microscopy systems. This work shows different ways to process the images which contain various blood phenomena happening in microfluidic devices and in microcirculation. For this purpose, the current methods used for tracking red blood cells (RBCs) flowing through a glass capillary and techniques to measure the cell-free layer thickness in different kinds of microchannels will be presented. Most of the past blood flow experimental data have been collected and analyzed by means of manual methods, that can be extremely reliable, but they are highly time-consuming, user-intensive, repetitive, and the results can be subjective to user-induced errors. For this reason, it is crucial to develop image analysis methods able to obtain the data automatically. Concerning automatic image analysis methods for individual RBCs tracking and to measure the well known microfluidic phenomena cell-free layer, two developed methods are presented and discussed in order to demonstrate their feasibility to obtain accurate data acquisition in such studies. Additionally, a comparison analysis between manual and automatic methods was performed.

4K 3-dimensional video microscope system (orbeye) for transsphenoidal pituitary surgery

BackgroundIn previous reports on experiences with an exoscope, this new technology was not found to be applicable for transsphenoidal pituitary surgery. As a specialized center for pituitary surgery, we were using a 4K 3D video microscope (Orbeye, Olympus) to evaluate the system for its use in transsphenoidal pituitary surgery in comparison to conventional microscopy.MethodWe report on 296 cases performed with the Orbeye at a single institution. An observational study was conducted with standardized subjective evaluation by the surgeons after each procedure. An objective measurement was added to compare the exoscopic and microscopic methods, involving surgery time and the initial postoperative remission rate in matched cohorts.ResultsThe patients presented with a wide range of pathologies. No serious events or minor complications occurred based on the usage of the 4K 3D exoscope. There was no need for switching back to the microscope in any of the cases. Compared to our microsurgically operated collective, there was no significant difference regarding duration of surgery, complications, or extent of resection. The surgeons rated the Orbeye beneficial in regard to instrument size, positioning, surgeon’s ergonomics, learning curve, image resolution, and high magnification.ConclusionsThe Orbeye exoscope presents with optical and digital zoom options as well as a 4K image resolution and 3D visualization resulting in better depth perception and flexibility in comparison to the microscope. Split screen mode offers the complementary benefit of the endoscope which may increase the possibilities of lateral view but has to be evaluated in comparison to endoscopic transsphenoidal procedures in the next step.

Bubbles Moving in Blood Flow in a Microchannel Network: The Effect on the Local Hematocrit.

Air inside of blood vessels is a phenomenon known as gas embolism. During the past years, studies have been performed to assess the influence of air bubbles in microcirculation. In this study, we investigated the flow of bubbles in a microchannel network with several bifurcations, mimicking part of a capillary system. Thus, two working fluids were used, composed by sheep red blood cells (RBCs) suspended in a Dextran 40 solution with different hematocrits (5% and 10%). The experiments were carried out in a polydimethylsiloxane (PDMS) microchannel network fabricated by a soft lithography. A high-speed video microscopy system was used to obtain the results for a blood flow rate of 10 µL/min. This system enables the visualization of bubble formation and flow along the network. The results showed that the passage of air bubbles strongly influences the cell’s local concentration, since a higher concentration of cells was observed upstream of the bubble, whereas a lower local hematocrit was visualized at the region downstream of the bubble. In bifurcations, bubbles may split asymmetrically, leading to an uneven distribution of RBCs between the outflow branches.

A Microfluidic Deformability Assessment of Pathological Red Blood Cells Flowing in a Hyperbolic Converging Microchannel.

The loss of the red blood cells (RBCs) deformability is related with many human diseases, such as malaria, hereditary spherocytosis, sickle cell disease, or renal diseases. Hence, during the last years, a variety of technologies have been proposed to gain insights into the factors affecting the RBCs deformability and their possible direct association with several blood pathologies. In this work, we present a simple microfluidic tool that provides the assessment of motions and deformations of RBCs of end-stage kidney disease (ESKD) patients, under a well-controlled microenvironment. All of the flow studies were performed within a hyperbolic converging microchannels where single-cell deformability was assessed under a controlled homogeneous extensional flow field. By using a passive microfluidic device, RBCs passing through a hyperbolic-shaped contraction were measured by a high-speed video microscopy system, and the velocities and deformability ratios (DR) calculated. Blood samples from 27 individuals, including seven healthy controls and 20 having ESKD with or without diabetes, were analysed. The obtained data indicates that the proposed device is able to detect changes in DR of the RBCs, allowing for distinguishing the samples from the healthy controls and the patients. Overall, the deformability of ESKD patients with and without diabetes type II is lower in comparison with the RBCs from the healthy controls, with this difference being more evident for the group of ESKD patients with diabetes. RBCs from ESKD patients without diabetes elongate on average 8% less, within the hyperbolic contraction, as compared to healthy controls; whereas, RBCs from ESKD patients with diabetes elongate on average 14% less than the healthy controls. The proposed strategy can be easily transformed into a simple and inexpensive diagnostic microfluidic system to assess blood cells deformability due to the huge progress in image processing and high-speed microvisualization technology.

Fire-shaped cylindrical glass micronozzles to measure cell deformability

Most blood cells exhibit a natural deformation capability. Specifically, red blood cells (RBCs) are characterized by their great ability to deform and squeeze through microcapillary vessels, even with strict restrictions, recovering their shape after crossing them. That capability can be affected by various blood diseases (malaria, diabetes, etc), turning the cells into more rigid, and, as a result, it can generate blood flow disorders in the microcirculatory system. In this work, we propose a new micro-device to study the cell deformability consisting of a simple borosilicate micronozzle whose production is straightforward, fast, and uses low-cost equipment, when compared with other similar microfluidic devices found in the literature. We checked that the optical distortion coming from the glass is negligible when the micronozzle was submerged in a bath of glycerol, being able to perform blood flow visualizations through the micronozzle by using a high-speed video microscopy system. The shape of the converging area of our micronozzles produced an extensional flow. This type of flow is proper to conduct cell deformability assessments. After the characterization of our micro-device, we applied it to obtain the deformation index (DI) of both healthy and chemically treated human RBCs for several sections of our micronozzles. As expected, the experimental results show that the DI of healthy RBCs is much higher than the corresponding for more rigid (chemically treated) ones. These measurements show that the borosilicate nozzles allow to detect changes in the cellular mechanical properties similar to those produced by blood diseases, and suggest that, this microfluidic device could be easily transformed into a clinical tool for detection of blood diseases.

In vitro blood flow visualizations and cell-free layer (CFL) measurements in a microchannel network

Microvascular networks are not simple straight microchannels but rather complex geometries composed by successive asymmetric divergent and convergent bifurcations. Despite the extensive research work in this field, still lack of knowledge about the blood flow behavior in microvascular networks. The current study applies the most current advanced visualization and microfabrication techniques to provide further insights into to the blood flow in network geometries. Hence, by using a high-speed video microscopy system, blood flow measurements and visualizations of the cell-free layer (CFL) were performed along a microchannel network composed by several divergent and convergent bifurcations. The inlet flow rate was kept constant whereas the hematocrit (Hct) and the depth of the geometry was changed in order to evaluate their effects into the CFL thickness. The results, show clearly that the Hct has a significant impact on the CFL thickness whereas the effect of reducing the depth did not contribute to a noticeable change on the CFL. In addition, the in vitro blood flow results reported here provide for the first time that in microfluidic devices having several asymmetric confluences it is likely to have the formation of several CFLs not only around the walls but also in middle of the main channels just downstream of the last confluence apex. Although, to best of our knowledge there is no evidence that this kind of flow phenomenon also happens in vivo, we believe that for microvascular networks with similar geometries and under similar flow conditions tested in this work, this kind of phenomenon may also happen in vivo. Furthermore, the results from this study could be extremely helpful to validate current numerical microvascular network models and to develop more realistic multiphase numerical models of blood flow in microcirculation.

Inflammation Increases the Opening State of Channels in the Epithelium, Releasing High Levels of ATP that Resulted in a Decrease on Mucociliary Transport Velocity in the Airways

ATP is one of the chemical signals locally released by the airway epithelium associated with an autocrine/paracrine mechanism to control basal ciliary beat frequency (CBF). A recent study has reported that ATP has a dual effect on CBF, since ATP (μM) increases CBF, but high levels of ATP (mM) significantly decreases CBF in cultures of mouse tracheal epithelial cell. This nucleotide can be released by different mechanisms that include hemichannels of connexins (Cx43) and pannexins (Panx1), and more recently the Calhm1 channel. In several cell types it has recently reported that in pathophysiological conditions, connexins and pannexins channels could modify their opening state. Furthermore, high levels of ATP have been measured in the bronchoalveolar fluid of asthmatic patients and ovoalbumin‐induced asthmatic mouse model (OVA). The asthmatic patients also have infiltration of inflammatory cells in the pulmonary epithelium, reduced CBF and mucociliary transport effectiveness. Mucociliary transport is the first defense mechanism in the airways, where particles are trapped in the mucus and removed by the ciliary beating to the pharynx. We proposed the following objective: Determine the relation between levels of ATP of an asthmatic OVA mouse model, the opening state of the channels that control the release of this nucleotide and the effectiveness of mucociliary transport in the airways. The protocols used in this study were approved, by the ethics committee of the university that follow international guidelines for animal care. We measured extracellular ([ATP]e) with a luminometric assay and evaluated the infiltration of inflammatory cells in the lung parenchyma by histochemistry (H&E). The ethidium bromide (5μM) capture was recorded with a camera mounted on an Olympus upright microscope. The microspheres (6μm) velocity on the surface of the trachea epithelium was registered with a videomicroscopy system and analyzed with the ImageJ program. We observed greater infiltration of inflammatory cells in the pulmonary parenchyma of the asthmatic mouse, compared with the control mouse. Also, we established that high levels of ATP were present in asthmatic mice trachea lavage compared to control animals. The microspheres velocity was diminished in OVA mouse model compare to control mice and addition of 1mM of ATP in control or asthmatic mice transiently stopped the microspheres movement. Furthermore, we observed a significant increase in the percentage of ethidium bromide capture by the epithelium of asthmatic mice compare to controls. These results demonstrated that in an inflammatory condition, channels involved in the ATP release increased their opening state in the airways epithelium. This effect could be associated to high levels of ATP measured in asthma, condition that resulted in a deleterious effect on mucociliary transport effectiveness.Support or Funding InformationCONICYT 21160416, LL.A. Millennium Institute on Immunology and Immunotherapy (P09/016‐F) MV.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Is regulation of microglial functions by T3 dependent on the micro‐environment?: Insights from the brainstem respiratory control network of newborn mice

We previously showed that thyroid hormones (THs) facilitate respiratory control development in newborn rodents. To understand the underlying mechanisms, we tested the hypotheses that TH, more precisely T3, 1) influences microglial functions in the brainstem of newborn mice and 2) this effect is region dependent. Measurements were performed on primary cultured mouse microglia isolated from the mixed glial cultures of postnatal day 3 C57BL/6J mice. Primary cultured neurons from cortex and brainstem were obtained from embryonic day 14–16 C57BL/6J mice. Microglia from cortex and brainstem were either cultured alone (DMEM) or co‐cultured with respective neurons (DMEM + neurobasal medium) under standard conditions (37°C, 5% CO2 / 90% humidity). Microglial motility (μm traveled) was monitored using a time‐lapse video‐microscopy system over 3h with or without T3 (1μM). Phagocytosis was quantified and observed in brainstem microglial cells pre‐incubated with 54 μg/ml Dextran, Rhodamine B (10 000 MW, neutral) in DMEM for 1h followed by incubation in DMEM with or without T3 (1 μM) for 3h. The cells were then fixed by 4% PFA. In the presence of neurons, but not in single culture, exposure to T3 increased brainstem microglial motility by 350% relative to control over the 3 h period (13.0 ± 4.5 vs 45.2 ± 7.8 μm; P=0.004). Exposing cortex cells to T3 also increased microglial motility; however, the effect was less important (156%; 46.0 ± 4.9 vs 72.1 ± 8.8 μm; P=0.009). The region of origin could also influence microglial functions in co‐culture as brainstem microglia showed decreased motility compared to cortex microglia (with and without T3). By comparison with controls, brainstem microglial cells cultured alone responded to T3 exposure by a ~4‐fold increase in phagocytosis as indicated by the fluorescence intensity of Dextran Rhodamine B (average arbitrary unit/area; 5.4 ± 1.1 vs 1.3 ± 0.2 AU/μm2; P=0.001). We conclude that 1) T3 facilitates multiple functional responses of microglia including motility and phagocytosis; both functions are important in regulation of synaptic function during development. 2) Microglial activation is specific to the surrounding environment, such as presence of thyroid hormones and brain regions. Microglia therefore appear as important effectors of TH action in the development of the respiratory network. Understanding the mechanisms by which these cells influence the generation and regulation of respiratory activity offers new approaches to alleviate respiratory disorders in newborn, especially those born pre‐term.Support or Funding InformationSupported by NSERC, Fonds de Recherche en Santé du Québec and Research Support Center, Graduate School of Medical Sciences, Kyushu University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Patient-Side Appraisal of Late Radiation-Induced Oral Microvascular Changes

Todetermine the clinical feasibility of examining and measuring late irradiation changes in the oral microcirculation of head and neck (HN) cancer patients using the novel CytoCam video microscope system. In 30 HN cancer patients and 30 age-matched controls, bilateral video images were recorded noninvasively of the oral microcirculation of the buccal mucosa and mandibular gingiva. Tissue perfusion parameters, such as functional capillary density (FCD), buccal blood vessel diameter, and microcirculatory flow index, were analyzed. No difference was observed for mean buccal mucosa FCD in irradiated versus healthy tissue, whereas a lower mean gingival FCD in irradiated versus healthy tissue was observed (34±17 capillaries per millimeter squared [cpll/mm2] vs 68±19 cpll/mm2; P<.001). A significant difference in mean buccal blood vessel diameter of 16±3μm was measured, compared with 14±1μm in control buccal mucosa(P<.001). No significant difference in microcirculatory flow index was observed between the 2 groups. Quantifying oral microcirculatory injury associated with late irradiation effects using the CytoCam was feasible in HN cancer patients. Results indicate that marked differences in tissue-specific microcirculatory measurements of angioarchitecture, diminished capillary density, and extensively dilated blood vessel diameters are associated with late irradiation effects in HN cancer patients.

Cell-free layer measurements of in vitro blood flow in a microfluidic network: an automatic and manual approach

ABSTRACTIn microcirculation, the cell-free layer (CFL) is a well-known physiological phenomenon that plays an important role in reducing the flow resistance and in balancing nitric oxide (NO) production by endothelial cells and NO scavenging by red blood cells. To better understand this phenomenon, several blood flow studies have been performed in simple geometries at both in vivo and in vitro environments. However, to date little information is available regarding the effects imposed by a complex branching network on the CFL. The present study shows the CFL layer variation at a microchannel network. The images were captured using a high-speed video microscopy system and the thickness of the CFL was measured using both manual and automatic image analysis techniques. Using this methodology, it was possible to visualise the in vitro blood flowing through the network and to identify several flow phenomena that happen in microcirculation. Overall, the results have shown that the concentration of cells and the geometrical configuration of the network have a major impact on the CFL thickness. In particular, the thickness of the CFL decreases as the fluid flows through a microchannel network composed with successive smaller channels. It was also clear that, for the full length of the network, the CFL thickness tends to decrease with the increase of the concentration of cells. The automatic method developed becomes inaccurate for high haematocrit and needs be calibrated by manual methods for Hcts bigger than 10%. The results obtained from this study could help the development and validation of multiscale numerical models able to take into account the CFL for simulating microvascular blood flow.

Removing the effect of blooming from potential energy measurement by employing total internal reflection microscopy integrated with video microscopy

Total internal reflection microscopy (TIRM) measures the interactions between a colloidal particle and a flat surface in aqueous solution. Recently, TIRM has further integrated with video microscopy (VM) and enabled the simultaneous measurements of multi-particle colloid-surface interactions in the same ensemble. However, there still remain challenges about accurate image acquisition due to blooming. Blooming means the number of photons reaching the detector exceeds its maximum capacity, and the excess photons will either spill to adjacent pixels or not be counted, leading to an obstacle from precise determination of intensity. Our result shows that blooming gives rise to a deviation of the measured potential energy from the classical theory of Derjaguin, Landau, Verway, and Overbeek (DLVO). Therefore, a correction method was developed in this work to deduce the real intensity from the experimental measurement. The relationship between scattered light intensity and exposure time deviates from linearity when blooming occurs. A correction equation was developed to recover the real intensity, which was then confirmed by the accordance between the corresponding potential energy profiles and the DLVO theory. This correction method is suitable for VM systems of colloidal probes illuminated by scattered light, broadening the application of VM imaging to investigate colloidal interactions.