Fluorescence Video Research Articles

Plasma Biomarkers Of Glycocalyx Shedding After Hemorrhage And Resuscitation: Correlation With Microvascular Events

The microvascular endothelial glycocalyx plays an essential role in several physiological functions but can be damaged after hemorrhage and fluid resuscitation. Many studies report plasma levels of syndecan‐1 and heparansulfate as indirect markers of glycocalyx degradation. However, it is unknown how much these measures are representative of the changes occurring at microvascular level. To fill this void, we studied quantitatively the relationship between plasma biomarkers and changes in various microvascular parameters, including glycocalyx thickness, using hemorrhage and a wide range of resuscitation fluids. Rats were bled 40% of estimated total blood volume and resuscitated with different fluids(fresh whole blood, packed red blood cells:lactated Ringer's, fresh frozen plasma, 5% albumin, lactated Ringer's, 3% hypertonic sodium chloride solution, and normal saline). Control animal groups were either not hemorrhaged or not resuscitated. Intravital fluorescence video microscopy was used to estimate glycocalyx thickness and permeability in 270 post‐capillary venules from 58 cremaster preparations in 9 animal groups while other microvascular parameters were measured using non‐invasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. In hemorrhaged rats, mean arterial pressure decreased from 100 ± 2 mmHg to 50 ± 3 mmHg after bleeding, and remained at intensities significantly lower than baseline after resuscitation. Exposure of animals to hemorrhage and different resuscitation fluids resulted in various glycocalyx degradation levels as expressed by different levels of glycocalyx thickness and plasma concentrations of syndecan‐1 and heparan sulfate. Glycocalyx thickness changes were correlated with plasma level changes of syndecan‐1 (r = −0.9, p<0.01) and heparansulfate (r = −0.9, p<0.01). Microvascular permeability changes were correlated with changes in both plasma biomarkers (r = 0.7, p<0.05). Syndecan‐1 and heparan sulfate were also correlated (r = 0.7, p<0.05). Except for diameter and permeability, changes in microvascular parameters (red blood cell velocity, blood flow, wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. Concurrent measurements of microvascular parameters, glycocalyx thickness and levels of plasma biomarkers, coupled with evaluation of systemic parameters, allowed a comprehensive evaluation of glycocalyx degradation after hemorrhage and resuscitation. The quantitative framework provided here suggests that monitoring plasma biomarkers of glycocalyx shedding may be useful in guiding resuscitation strategies following hemorrhage.Support or Funding InformationSupported by US Army Medical Research & Materiel Command.

Techniques to Distinguish Apoptosis from Necroptosis.

The processes by which cells die are as tightly regulated as those that govern cell growth and proliferation. Recent studies of the molecular pathways that regulate and execute cell death have uncovered a plethora of signaling cascades that lead to distinct modes of cell death, including "apoptosis," "necrosis," "autophagic cell death," and "mitotic catastrophe." Cells can readily switch from one form of death to another; therefore, it is vital to have the ability to monitor the form of death that cells are undergoing. A number of techniques are available that allow the detection of cell death and when combined with either knockdown approaches or inhibitors of specific signaling pathways, such as caspase or RIP kinase pathways, they allow the rapid dissection of divergent cell death pathways. However, techniques that reveal the end point of cell death cannot reconstruct the sequence of events that have led to death; therefore, they need to be complemented with methods that can distinguish all forms of cell death. Apoptotic cells frequently undergo secondary necrosis under in vitro culture conditions; therefore, novel methods relying on high-throughput time-lapse fluorescence video microscopy are necessary to provide temporal resolution to cell death events. Further, visualizing the assembly of multiprotein signaling hubs that can execute apoptosis or necroptosis helps to explore the underlying processes. Here we introduce a suite of techniques that reliably distinguish necrosis from apoptosis and secondary necrosis, and that enable investigation of signaling platforms capable of instructing apoptosis or necroptosis.

The Role of Neuroendoscope with Fluorescence Angiography in the Aneurysmal Surgery Located in the Skull Base Lesion

Background: The neuroendoscope, with its higher magnification, better observation, and additional illumination, can provide us information that may not be available with the microscope, and is exceedingly useful in aneurysm surgery, especially located in the skull base lesion, for visualizing structures hidden in the dead angle. However, ordinary endoscopy cannot provide real-time assessment of arterial flow, which is essential after clip placement to avoid complications. To overcome this shortcoming, we have introduced a newly-developed technique of intraoperative endoscopic fluorescence video angiography. In this study, we analyzed the roles of this novel technique for aneurysm surgery located in the skull base lesion.

Evaluation of luminosity of subpixels on a luminescent monitor based on CdSe/CdS/ZnS quantum dots

Modern advances in the synthesis of new materials based on semiconductor CdSe/CdS/ZnS quantum dots have yielded phosphors with a unique set of physical, optical, and surface properties. The high degree of compatibility of quantum dots with some polymers allows one to obtain highly stable polymeric fluorescent layers excited by radiation at a wavelength in the blue region of the visible range. The elaboration of highly efficient and low-cost blue LEDs by Japanese researchers has enabled us to consider the use of these materials for new-generation energy-saving fluorescent screens and video monitors. In this paper we discuss the basic laws of the collimated beam passing through the excitation of the fluorescent material layer and examine the conditions for the highest possible visual brightness of subpixels and the maximum color gamut of the image.

Single Cell Analysis of a Bacterial Sender-Receiver System.

Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined.

COPI selectively drives maturation of the early Golgi.

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.

Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination.

The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin-resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence-based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live-cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase-3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin.

Analysis-preserving video microscopy compression via correlation and mathematical morphology.

The large amount video data produced by multi-channel, high-resolution microscopy system drives the need for a new high-performance domain-specific video compression technique. We describe a novel compression method for video microscopy data. The method is based on Pearson's correlation and mathematical morphology. The method makes use of the point-spread function (PSF) in the microscopy video acquisition phase. We compare our method to other lossless compression methods and to lossy JPEG, JPEG2000, and H.264 compression for various kinds of video microscopy data including fluorescence video and brightfield video. We find that for certain data sets, the new method compresses much better than lossless compression with no impact on analysis results. It achieved a best compressed size of 0.77% of the original size, 25× smaller than the best lossless technique (which yields 20% for the same video). The compressed size scales with the video's scientific data content. Further testing showed that existing lossy algorithms greatly impacted data analysis at similar compression sizes.

Logarithmic intensity compression in fluorescence guided surgery applications.

The use of fluorescence video imaging to guide surgery is rapidly expanding, and improvements in camera readout dynamic range have not matched display capabilities. Logarithmic intensity compression is a fast, single-step mapping technique that can map the useable dynamic range of high-bit fluorescence images onto the typical 8-bit display and potentially be a variable dynamic contrast enhancement tool. We demonstrate a ∼4.6 times improvement in image quality quantified by image entropy and a dynamic range reduction by a factor of ∼380 by the use of log-compression tools in processing in vivo fluorescence images.

Structure and Strength of Flocs of Precipitated Calcium Carbonate Induced by Various Polymers Used in Papermaking

Because of persistent economic pressure on cost reduction, inorganic fillers such as precipitated calcium carbonate (PCC) have become increasingly economically attractive in the papermaking process. The increase of filler level in paper can be achieved by adding it to pulp prior to the headbox, either as individual filler particles or as preaggregates, while maintaining paper strength and minimizing their negative impact. Consequently, the floc structure and strength of PCC aggregates was studied using flocculants and dry strength agents, using static light scattering/diffraction (SLS), real time fluorescent video imaging (RTFVI), image analysis, photometric dispersion analysis (PDA), and scanning electron microscopy (SEM). It was found that PEO/cofactor induced PCC aggregates were weaker at high shear and far more irreversible than those induced by the partially hydrolyzed polyvinyl formamide copolymerized with acrylic acid (PVFA/NaAA) or cationic starch. Flocs produced at low polymer dosages were smalle...

April 2015: This Month in JoVE - Studying Locomotion in Drunken Worms, Preserving Human Liver for Transplantation, and Visualizing Bacterial Swarms

Here's a look at what's coming up in the April 2015 issue of JoVE: The Journal of Visualized Experiments. In JoVE Neuroscience, it is clear that alcohol has profound effects on the brain. But why do different individuals respond so differently to alcohol, and why are some more prone to abuse? Some of the answers lie in our genes. In the nematode worm C. elegans, genes are easy to manipulate, and the neurobiology is simple. So it's an excellent model to study the effects of alcohol on behavior. Davies et al. use copper rings to corral worms with different genotypes on agar plates containing alcohol. They take videos of the worms at set time points and measure the locomotion of each genotype using object recognition software With this procedure, C. elegans can help unravel the molecular basis of alcohol sensitivity and tolerance, as well as the risk of abuse. In JoVE Medicine, alcohol doesn't just affect the brain; it also affects the liver, and for many patients with alcoholic liver disease, liver transplantation is the only hope. But there is a severe shortage of livers available for transplants. Therefore, Bruinsma et al. present an alternative way to preserve donor livers, which are traditionally stored statically on ice. In this new method, the donor liver is kept alive by a perfusion machine and perfused at 21°C-warmer than ice, but lower than normal body temperatures, which allows the liver to be assessed for function and transplantability. This technique may greatly expand the pool of donor livers available to patients in need of transplants. In JoVE Biology, one of the most commonly studied modes of bacterial motility is called swarming. However, the reproducibility of swarm plate assays can be difficult to achieve, so Morales-Soto et al. present a method that standardizes parameters (such as moisture content of plates) that affect swarming motility. By using bacterial strains expressing different fluorescent proteins, they can observe swarming competition dynamics between different species in a fluorescent time-lapsed video. This method can help elucidate various aspects of bacterial growth, such as how bacteria colonize different types of surfaces. In JoVE Behavior, Labots et al. demonstrate the modified hole board. This behavioral testing apparatus consists of a protected box and a board containing small cylinders. In one type of experiment, the board can be baited with tasty food rewards. In some cylinders, the treat is locked under a grid, and in differently colored cylinders, the treat is accessible. Small laboratory mammals (such as rats) are conditioned to the food reward and tested for behaviors such as object recognition or food intake inhibition. This test can help measure unconditioned behavior of small laboratory mammals and analyze their cognitive abilities. You've just had a sneak peek of the April 2015 issue of JoVE. Visit the website to see the full-length articles, plus many more, in JoVE: The Journal of Visualized Experiments.

Microvascular Responses to Ischemic Stroke following Splenectomy

Splenectomy is known to protect the brain against the tissue injury that results from ischemia and reperfusion (I/R). The mechanisms that underlie this neuroprotective effect of splenectomy remain poorly understood. An early response to brain I/R is the recruitment of inflammatory cells and platelets into the cerebral microvasculature. The objective of this study was to determine if splenectomy alters the recruitment of adherent leukocytes and platelets in cerebral microvessels in the early phase (24 hr) of reperfusion following ischemic stroke. Wild type C57Bl/6 mice underwent splenectomy or sham splenectomy two weeks prior to 45 min middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. After 24 h of reperfusion, intravital fluorescence video microscopy was used to monitor and quantify the adhesion of leukocytes (rhodamine‐G6 labeled) and platelets (CFSE‐labeled) in cerebral venules. Infarct volume and peripheral blood counts were also measured. Splenectomy attenuates the recruitment of adherent platelets and leukocytes in the cerebral microvasculature and reduces infarct volume. The increased blood platelet count observed following MCAO was reduced to control levels in splenectomized animals. Peripheral leukocyte count did not differ between the splenectomized and sham splenectomized mice. These results implicate the spleen in I/R‐induced brain inflammation, thrombogenesis, and tissue necrosis as early as 24 h after reperfusion (Supported by a Malcolm Feist Postdoctoral Fellowship)

Functional characterization of Na+/H+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Quantitative assessment of the free jejunal graft perfusion

BackgroundReconstruction with free jejunal graft (FJG) is often performed for patients with hypopharyngeal or cervical esophageal cancer. During reconstruction with an FJG after pharyngoesophagectomy, it is critical to intraoperatively detect venous anastomotic failure and subsequent venous malperfusion to avoid postoperative FJG necrosis. This study introduces a novel method for assessing blood perfusion in FJGs by using indocyanine green (ICG) fluorescence angiography. MethodsWe used ICG fluorescence angiography to quantitatively assess FJG blood perfusion in archived fluorescence video files from 26 patients who had undergone FJG transfer. A software program “ROIs”, was used to create a time-fluorescence intensity curve. We retrospectively measured the maximum fluorescence intensity at the terminal ileum and the duration (T1/2max) between when the intensity began rising and when it reached half of the maximum. ResultsAmong the 26 patients, 5 patients suffered venous anastomotic failure. In three of these cases, anastomosis was corrected intraoperatively; the other two patients underwent a second FJG transfer. Retrospective assessment showed that the mean T1/2max at the FJG serosae was significantly longer in these five patients than that in FJGs with good blood perfusion. Our analysis revealed that a T1/2max >9.6 s may be a good indicator of FJG venous malperfusion. ConclusionsQuantitative analysis of ICG fluorescence angiography proved useful for detecting venous anastomotic failure of FJG, and may help to reduce vascular problems in FJG reconstruction.

Domestication of the green alga Chlorella sorokiniana: reduction of antenna size improves light-use efficiency in a photobioreactor.

BackgroundThe utilization of biomass from microalgae for biofuel production is one of the key elements for the development of a sustainable and secure energy supply. Among the different microalgae, Chlorella species are of interest because of their high productivity, high lipid content, and resistance to the high light conditions typical of photobioreactors. However, the economic feasibility of growing algae at an industrial scale is yet to be realized, in part because of biological constraints that limit biomass yield. A key issue is the inefficient use of light due to uneven light distribution, and the dissipation of excess absorbed light as heat. The successful implementation of biofuel production facilities requires the development of algal strains with enhanced light use efficiency in photobioreactors. Such domestication strategies include decreasing the absorption cross section in order to enhance light penetration, increasing the size of metabolic sinks per chlorophyll and minimizing feedback energy dissipation.ResultsIn this work we applied random mutagenesis and phenotypic selection to the thermotolerant, fast-growing Chlorella species, C. sorokiniana. Truncated antenna mutants (TAMs) were selected that exhibited a lower fluorescence yield than the wild-type (WT) strain. Six putatively interesting mutants were selected by high throughput fluorescence video imaging, two of which, TAM-2 and TAM-4, were found to have approximately half the chlorophyll content per cell and LHCII complement per PSII with respect to the WT. In batch culture, TAM-2 showed an increased photon use efficiency, yielding a higher Pmax at saturating irradiances with respect to the WT. Cultivation of TAM-2 in both laboratory-scale and outdoor photobioreactors showed higher productivity than WT, with a 30% higher biomass yield in dense cell suspensions typical of industrial photobioreactors.ConclusionsThese results suggest that generation of mutants with low chlorophyll content can significantly improve the light-to-biomass conversion efficiency of C. sorokiniana under mass culture conditions. However, owing to the lack of sexual reproduction in this species, the presence of additional mutations might affect growth rate, suggesting that selection should include evaluation of multiple independent mutants for each desired phenotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0157-z) contains supplementary material, which is available to authorized users.

Abstract 3599: Long-distance intercellular transport of microRNAs via tunneling nanotubes: Role in tumor-stroma interactions and malignant potential

Abstract Introduction: Intercellular communication is vital to cancer cell organization and invasion. Identification of key cellular cargo and their preferred modes of transport is important, and yet it remains underdeveloped. Gap junctions and exosomes are known to play a key role as conduits in mediating intercellular transport. Tunneling nanotubes (TnTs) - long narrow actin-based cytoplasmic extensions - are unique structures that facilitate direct long distance cell-to-cell transport of cargo, potentially including microRNAs (miRNAs). These small non-coding RNAs play a significant role in pathogenesis, primarily in post-transcriptional gene regulation. We hypothesized that miRNA transport via TnTs occurs between malignant and stromal cells and can lead to changes in gene regulation that propagate the cancer phenotype. In this study, we used murine osteosarcoma and human ovarian cancer as model systems to investigate tumor-tumor and tumor-stroma interactions via TnTs. Methods: miR-19a was labelled with the fluorophore Alexa-488 by in vitro transcription, and visualized using confocal fluorescent video microscopy. K7M2 (murine osteosarcoma) cells were transfected with Alexa-488 labeled miR-19a prior to co-culture with K7M2 or MC3T3 murine osteoblast cells. Parallel studies were performed using miR-199a in human ovarian cancer (SKOV3 and C200) and normal human ovarian epithelial cells (IOSE). Movement of miRNAs was imaged by time-lapse microscopy every 5 minutes up to 48 hours. Intercellular transport of miRNAs was further studied by co-culture of cells separated by a membrane filter with pores 0.4 µm in size in a transwell chamber, which is restrictive of cell migration and exosome transfer but permissive of TnT formation. Cells were collected at 48 hours and relative miR-19a levels were assessed in recipient cells using qPCR. Results: TnT-mediated cell-to-cell transport of miR-19a was demonstrated among K7M2 cells themselves and between K7M2 and MC3T3 cells using fluorescence time-lapse imaging. miRNA transport was detected in TnTs spanning 40-200 µm in length, occurring over 25 minutes to several hours. Relative endogenous levels of miR-19a were elevated up to 2.4-fold in MC3T3 osteoblast cells after 48 hours of co-culture with osteosarcoma cells. Similar experiments performed in human ovarian cancer and ovarian epithelial cells also confirmed transport of miR-199a between malignant and stromal cells. Conclusions: To our knowledge, we provide the first demonstration of intercellular transport of miRNAs between cancer and stromal cells via TnTs. This discovery establishes a novel mechanism by which miRNAs mediate altered gene regulation in the complex and heterogeneous tumor microenvironment. We are further investigating the effects of TnT-mediated miRNA transfer on recipient stromal cells and its effects on the invasive phenotype of solid tumors. Citation Format: Venugopal Thayanithy, Elizabeth L. Dickson, Subbaya Subramanian, Clifford J. Steer, Emil Lou. Long-distance intercellular transport of microRNAs via tunneling nanotubes: Role in tumor-stroma interactions and malignant potential. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3599. doi:10.1158/1538-7445.AM2014-3599

Effect of bending flexibility on the phase behavior and dynamics of rods.

We study by means of molecular and Brownian dynamics simulations the influence of bending flexibility on the phase behavior and dynamics of monodisperse hard filamentous particles with an aspect ratio of 8 and persistence lengths equal to 3 and 11 times the particle length. Although our particles are much shorter, the latter corresponds to the values for wild-type and mutant fd virus particles that have been subject of a recent experimental study, where the diffusion of these particles in the nematic and smectic-A phase was investigated by means of video fluorescence microscopy [E. Pouget, E. Grelet, and M. P. Lettinga, Phys. Rev. E 84, 041704 (2011)]. In agreement with theoretical predictions and simulations, we find that for the more flexible particles (shorter persistence length) the nematic (N) to smectic-A (Sm-A) phase transition shifts to larger values of the particle density. Interestingly, we find that for the more rigid particles (larger persistence length), the smectic layer-to-layer distance decreases monotonically with increasing density, whereas for the more flexible ones, it first increases, reaches a maximum and then decreases. For our more flexible particles, we find a smectic-B phase at sufficiently high densities. Moreover, in line with experimental observations and theoretical predictions, we find heterogeneous dynamics in the Sm-A phase, in which particles hop between the smectic layers. We compare the diffusion of our two types of particle at identical values of smectic order parameter, and find that flexibility does not change the diffusive behavior of particles along the director yet significantly slows down the diffusion perpendicular to it. In our simulations, the ratio of diffusion constants along and perpendicular to the director decreases just beyond the N-Sm-A phase transition for both our stiff and more flexible particles.

Intraoperative Laser-Assisted Indocyanine Green Imaging for Objective Measurement of the Vascular Delay Technique in Locoregional Head and Neck Flaps

Reconstruction of oncologic or traumatic head and neck defects often requires complex planning of locoregional, pedicled, or interpolated flaps. In cases with a higher risk of flap failure, vascular delay with staged reconstruction can help improve tissue perfusion and increase chances of flap survival. An objective tool is needed to help guide reconstructive surgeons with the intraoperative decision to pursue vascular delay. To describe a pilot study using a novel application of a technique that quantifies and validates the benefit of the vascular delay procedure in locoregional flaps and to demonstrate a practical and broadly applicable technology that can be easily incorporated into intraoperative decision making and improve outcomes for high-risk flaps. A pilot study using intraoperative laser-assisted indocyanine green imaging measurements and fluorescence videos to objectively measure the benefit of vascular delay procedures in patients with head and neck defects and wound healing risk factors requiring locoregional flap reconstruction at an academic tertiary care center. Intraoperative laser-assisted indocyanine green imaging with video documentation and quantitative measurements was used to evaluate flap perfusion before a vascular delay procedure. Measurements were repeated after a 3-week vascular delay procedure. Two patients were identified based on comorbid conditions that resulted in a higher risk of flap failure, as well as the need for a locoregional flap for reconstruction. At the initial elevation of the flap, quantitative results from flap imaging demonstrated low perfusion numbers and minimal fluorescence, suggesting poor tissue perfusion and increased likelihood of postoperative flap compromise or failure. Following a vascular delay of 3 weeks, repeat measurements were substantially improved. No wound healing issues were observed. This is the first study to date to quantitatively demonstrate the benefit of the vascular delay technique in patients with potential vascular compromise in locoregional head and neck flap reconstruction. Data obtained suggest that this technology can be used to guide intraoperative decision making in complicated reconstructions and help optimize patient outcomes. NA.

Fluctuations of DNA mobility in nanofluidic entropic traps.

We studied the mobility of DNA molecules driven by an electric field through a nanofluidic device containing a periodic array of deep and shallow regions termed entropic traps. The mobility of a group of DNA molecules was measured by fluorescent video microscopy. Since the depth of a shallow region is smaller than the DNA equilibrium size, DNA molecules are trapped for a characteristic time and must compress themselves to traverse the boundary between deep and shallow regions. Consistent with previous experimental results, we observed a nonlinear relationship between the mobility and electric field strength, and that longer DNA molecules have larger mobility. In repeated measurements under seemingly identical conditions, we measured fluctuations in the mobility significantly larger than expected from statistical variation. The variation was more pronounced for lower electric field strengths where the trapping time is considerable relative to the drift time. To determine the origin of these fluctuations, we investigated the dependence of the mobility on several variables: DNA concentration, ionic strength of the solvent, fluorescent dye staining ratio, electroosmotic flow, and electric field strength. The mobility fluctuations were moderately enhanced in conditions of reduced ionic strength and electroosmotic flow.

Endothelial progenitor cells augment collateralization and hemodynamic rescue in a model of chronic cerebral ischemia.

Surgical flow augmentation for treatment of cerebral hemodynamic impairment remains controversial. Here, we investigated the benefit of endothelial progenitor cell (EPC) treatment in a rat model of chronic cerebral hypoperfusion. At repeated time points after 3-vessel occlusion (3-VO), animals were treated with 1 × 10(6) DiI-labeled (a) ex vivo-expanded embryonic-EPC (e-EPC), (b) cyclic AMP-differentiated embryonic-endothelial progenitor-derived cells (e-EPDC as biologic control) or, (c) saline. The cerebrovascular reserve capacity (CVRC) was assessed immediately before and on days 7 and 21 after 3-VO. Structural effects were assessed by latex perfusion, immunohistochemistry, and intravital fluorescence video microscopy on day 21. Three-vessel occlusion resulted in a significant impairment of the CVRC with better functional recovery after treatment with e-EPC (16.4±8%) compared with e-EPDC (3.7±8%) or saline (6.4±9%) by day 21 (P<0.05), which was paralleled by a significant increase in the vessel diameters of the anterior Circle of Willis, a significantly higher number of leptomeningeal anastomoses and higher parenchymal capillary density in e-EPC-treated animals. Interestingly, despite in vivo interaction of e-EPC with the cerebral endothelium, e-EPC incorporation into the cerebral vasculature was not observed. Our results suggest that EPC may serve as a novel therapeutic agent in clinical trials for nonsurgical treatment of chronic cerebral hemodynamic impairment.