Quantification of oxygen consumption in head and neck cancer using fluorescent sensor foil technology.

Introduction: Head and neck squamous cell carcinoma (HNSCC) patients suffer from frequent local recurrences that negatively impact on prognosis. Hence, distinguishing tumor and normal tissue is of clinical importance as it may improve the detection of residual tumor tissue in surgical resection margins and during imaging-based surgery planning. Differences in O2 consumption (OC) can be used to this aim, as they provide options for improved surgical, image-guided approaches. Methods: In the present study, the potential of a fluorescent sensor foil-based technology to quantify OC in HNSCC was evaluated in an in vitro 3D model and in situ in patients. Results: In vitro measurements of OC using hypopharyngeal and esophageal cell lines allowed a specific detection of tumor cell spheroids embedded together with cancer-associated fibroblasts in type I collagen extracellular matrix down to a diameter of 440 µm. Pre-surgery in situ measurements were conducted with a handheld recording device and sensor foils with an oxygen permeable membrane and immobilized O2-reactive fluorescent dyes. Lateral tongue carcinoma and carcinoma of the floor of the mouth were chosen for analysis owing to their facilitated accessibility. OC was evaluated over a time span of 60 seconds and was significantly higher in tumor tissue compared to healthy mucosa in the vicinity of the tumor. Discussion: Hence, OC quantification using fluorescent sensor foil-based technology is a relevant parameter for the differentiation of tumor tissue of the head and neck region and may support surgery planning.

Physiological oxygen measurements in vitro-Schrödinger's cat in 3D cell biology.

After the development of 3D cell culture methods in the middle of the last century and the plethora of data generated with this culture configuration up to date, it could be shown that a three-dimensional arrangement of cells in most of the cases leads to a more physiological behavior of the generated tissue. However, a major determinant for an organotypic function, namely, the dissolved oxygen concentration in the used in vitro-system, has been neglected in most of the studies. This is due to the fact that the oxygen measurement in the beginning was simply not feasible and, if so, disturbed the measurement and/or the in vitro-system itself. This is especially true for the meanwhile more widespread use of 3D culture systems. Therefore, the tissues analyzed by these techniques can be considered as the Schrödinger's cat in 3D cell biology. In this perspective paper we will outline how the measurement and, moreover, the regulation of the dissolved oxygen concentration in vitro-3D culture systems could be established at all and how it may be possible to determine the oxygen concentration in organoid cultures and the respiratory capacity via mito stress tests, especially in spheroids in the size range of a few hundred micrometers, under physiological culture conditions, without disturbances or stress induction in the system and in a high-throughput fashion. By this, such systems will help to more efficiently translate tissue engineering approaches into new in vitro-platforms for fundamental and applied research as well as preclinical safety testing and clinical applications.

O2-sensitive microcavity arrays: A new platform for oxygen measurements in 3D cell cultures.

Oxygen concentration plays a crucial role in (3D) cell culture. However, the oxygen content in vitro is usually not comparable to the in vivo situation, which is partly due to the fact that most experiments are performed under ambient atmosphere supplemented with 5% CO2, which can lead to hyperoxia. Cultivation under physiological conditions is necessary, but also fails to have suitable measurement methods, especially in 3D cell culture. Current oxygen measurement methods rely on global oxygen measurements (dish or well) and can only be performed in 2D cultures. In this paper, we describe a system that allows the determination of oxygen in 3D cell culture, especially in the microenvironment of single spheroids/organoids. For this purpose, microthermoforming was used to generate microcavity arrays from oxygen-sensitive polymer films. In these oxygen-sensitive microcavity arrays (sensor arrays), spheroids cannot only be generated but also cultivated further. In initial experiments we could show that the system is able to perform mitochondrial stress tests in spheroid cultures to characterize mitochondrial respiration in 3D. Thus, with the help of sensor arrays, it is possible to determine oxygen label-free and in real-time in the immediate microenvironment of spheroid cultures for the first time.

High-resolution spatiotemporal pHe and pO2 imaging in head and neck and oesophageal carcinoma cells.

BACKGROUND: pO2 and pH are physiological parameters relevant for different processes in health and disease, including wound healing and cancer progression. Head and neck squamous cell carcinomas (HNSCC) and oesophageal squamous cell carcinomas (ESCC) have a high rate of local recurrence that is partly related to treatment-resistant residual tumour cells. Hence, novel diagnostic tools are required to visualise potential residual tumour cells and thereby improve treatment outcome for HNSCC and ESCC patients. We developed a device to spatiotemporally measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) to distinguish HNSCC and ESCC cells from healthy cells in vitro, exploiting general metabolic differences between cancer cells and healthy cells. METHODS: OCR and ECAR were measured via a newly developed device named STO2p-Q (SpatioTemporal O2 and pH Quantification) using the VisiSens technology based on ratiometric fluorescence imaging, facilitating spatiotemporal resolution. Results were confirmed using extracellular flux analyses (Seahorse technology). RESULTS: STO2p-Q is described and used to measure OCR and ECAR in HNSCC and ESCC cell lines and normal fibroblast and epithelial cells as components of the tumour microenvironment. OCR measurements showed differences amongst HNSCC and ESCC cell lines and between HNSCC/ESCC and normal cells, which on average had lower OCR than HNSCC/ESCC cells. Both OCR and ECAR measurements were independently verified using the Seahorse technology. Additionally, using STO2p-Q, HNSCC/ESCC, and normal cells could be spatially resolved with a resolution in the low millimetre range. CONCLUSIONS: We developed a method to spatiotemporally measure OCR and ECAR of cells, which has many potential in vitro applications and lays the foundation for the development of novel diagnostic tools for the detection of cancerous tissue in HNSCC and ESCC patients in vivo.

Automated 3D Microphysiometry Facilitates High-Content and Highly Reproducible Oxygen Measurements within 3D Cell Culture Models.

Microphysiometry is a powerful technique to study metabolic parameters and detect changes to external stimuli. However, applying this technique for automated label-free and real-time measurements within cell-laden three-dimensional (3D) cell culture constructs remains a challenge. Herein, we present an entirely automated microphysiometry setup that combines needle-type microsensors with motorized sample and sensor positioning systems inside a standard tissue-culture incubator. The setup records dissolved oxygen as a metabolic parameter along the z-direction within cell-laden 3D constructs in a minimally invasive manner. The microphysiometry setup was applied to characterize the spatial oxygen distribution within thick cell-laden 3D constructs, study the time-dependent changes on the oxygen tension within 3D breast cancer models following a chemotherapeutic treatment, and identify kinetics and recovery effects after drug exposure over 5 weeks. Our data suggest that the microphysiometry setup enables highly reproducible measurements without human intervention, due to the high degree of automation and positional accuracy. The results demonstrate the applicability of the setup to provide valuable long-term insights into oxygenation within 3D models using minimally invasive, label-free, and entirely automated analysis methods.

A New Non-invasive Technique for Measuring 3D-Oxygen Gradients in Wells During Mammalian Cell Culture.

Oxygen tension plays an important role in overall cell function and fate, regulating gene expression, and cell differentiation. Although there is extensive literature available that supports the previous statement, little information is to be found about accurate O2 measurements during culture. In fact, O2 concentration at the cell layer during culture is commonly assumed to be equal to that of the incubator atmosphere. This assumption does not consider oxygen diffusion properties, cell type, cell density, media composition, time in culture nor height of the cell culture medium column. In this study, we developed a non-invasive, optical sensor foil-based technique suitable for measuring the 3D oxygen gradient that is formed during cell culture as a result of normal cell respiration. For this propose, we created a 3D printed ramp to which surface an oxygen optode sensor foil was attached. The ramps were positioned inside the culture wells of 24 well plate prior cell seeding. This set up in conjunction with the VisiSens TD camera system allows to investigate the oxygen gradient formation during culture. Cultivation was performed with three different initial cell densities of the cell line A549 that were seeded on the plate containing the ramps with the oxygen sensors. The O2 gradient obtained after 96 h of culture showed significantly lower O2 concentrations closer to the bottom of the well in high cell density cultures compared to that of lower cell density cultures. Furthermore, it was very interesting to observe that even with low cell density culture, oxygen concentration near the cell layer was lower than that of the incubator atmosphere. The obtained oxygen gradient after 96 h was used to calculate the oxygen consumption rate (OCR) of the A549 cells, and the obtained value of ~100 fmol/h/cell matches the OCR value already reported in the literature for this cell line. Moreover, we found our set up to be unique in its ability to measure oxygen gradient formation in several wells of a cell culture plate simultaneously and in a non-invasive manner.

Imaging of pH and pO2 gives insight in molecular processes of irradiated cells.

One of the major challenges in radiation therapy is the interference with tissue repair processes due to hypoxic characteristics and pH dysregulation. In this study, we present dual imaging of pH and oxygenation in vitro based on luminescent biocompatible sensor foils that allow studying the effects of irradiation on different cell types in culture. Different sensitivities of fibroblast and oral squamous carcinoma cells were observed by complementing oxygen and pH differences with proliferation assays. This study highlights especially the distinct role of oxygen after irradiation and the difference in proliferation processes of irradiated normal dermal cells in contrast to irradiated tumor cells.

Oxygen-distribution within 3-D collagen I hydrogels for bone tissue engineering.

Tissue engineering (TE) approaches typically envisage the structural and functional reconstitution of previously damaged tissue in situ. An adequate three-dimensional environment is therefore of fundamental importance for the designated cells associated to the scaffold material. The sufficient supply with nutrients and oxygen in vitro and in vivo mark thereby critical challenges of TE. In this study, we intended to analyse the level of locally dissolved oxygen within 3-D cell-loaded collagen I gels in vitro. For the analysis of the oxygen levels in situ, we employed an optical fibre-based micro sensor setup, as well as a camera supported non-invasive optical sensor foil based technique. These complementary analytical tools enable the identification, localization, and temporal follow-up investigation of specified regions of interest within TE constructs. Human adipose-derived mesenchymal stem cells (hAdMSCs) cultured in collagen I gels under normoxic conditions were analysed periodically and kinetically up to 70 days - thereby revealing dynamic changes of the level of dissolved oxygen inside the gel constructs. Dependent on the applied cell concentration, the in vitro oxygen concentration (cO2) within the gels reached physiological ranges (7-9%) after 21 days, or 35 days of culture. The minimal cO2 was measured after 35 days in vitro, featuring an oxygen level of 4.8 ±â€¯1.3%. Upon prolonged culture, a plateau-like status of the cO2 around 8-9% established, indicating a change in the physiological activity of the cells under investigation. The expression patterns of BCL2, CASP3 and MCM5 revealed significant differences among the proliferative and apoptotic stages of the cell-loaded samples at the investigated time points of 7 and 70 days in culture. In summary, these data show the temporary dynamic nature of the oxygen distribution in cell-loaded gel constructs. The applied technique is an ideal tool for the evaluation of multiple parameters affecting the oxygen distribution in vitro. We conclude that it takes 5 weeks for establishing an equilibrium of cO2. Levels reached in a 3-D gel construct are comparable with physiological oxygenation ranges in bone-associated tissues.

Plant-Sediment Interactions in Salt Marshes - An Optode Imaging Study of O2, pH, and CO 2 Gradients in the Rhizosphere.

In many wetland plants, belowground transport of O2 via aerenchyma tissue and subsequent O2 loss across root surfaces generates small oxic root zones at depth in the rhizosphere with important consequences for carbon and nutrient cycling. This study demonstrates how roots of the intertidal salt-marsh plant Spartina anglica affect not only O2, but also pH and CO2 dynamics, resulting in distinct gradients of O2, pH, and CO2 in the rhizosphere. A novel planar optode system (VisiSens TD®, PreSens GmbH) was used for taking high-resolution 2D-images of the O2, pH, and CO2 distribution around roots during alternating light-dark cycles. Belowground sediment oxygenation was detected in the immediate vicinity of the roots, resulting in oxic root zones with a 1.7 mm radius from the root surface. CO2 accumulated around the roots, reaching a concentration up to threefold higher than the background concentration, and generally affected a larger area within a radius of 12.6 mm from the root surface. This contributed to a lowering of pH by 0.6 units around the roots. The O2, pH, and CO2 distribution was recorded on the same individual roots over diurnal light cycles in order to investigate the interlinkage between sediment oxygenation and CO2 and pH patterns. In the rhizosphere, oxic root zones showed higher oxygen concentrations during illumination of the aboveground biomass. In darkness, intraspecific differences were observed, where some plants maintained oxic root zones in darkness, while others did not. However, the temporal variation in sediment oxygenation was not reflected in the temporal variations of pH and CO2 around the roots, which were unaffected by changing light conditions at all times. This demonstrates that plant-mediated sediment oxygenation fueling microbial decomposition and chemical oxidation has limited impact on the dynamics of pH and CO2 in S. anglica rhizospheres, which may in turn be controlled by other processes such as root respiration and root exudation.

Dynamics of oxygen and carbon dioxide in rhizospheres of Lobelia dortmanna - a planar optode study of belowground gas exchange between plants and sediment.

Root-mediated CO2 uptake, O2 release and their effects on O2 and CO2 dynamics in the rhizosphere of Lobelia dortmanna were investigated. Novel planar optode technology, imaging CO2 and O2 distribution around single roots, provided insights into the spatiotemporal patterns of gas exchange between roots, sediment and microbial community. In light, O2 release and CO2 uptake were pronounced, resulting in a distinct oxygenated zone (radius: c. 3 mm) and a CO2 -depleted zone (radius: c. 2 mm) around roots. Simultaneously, however, microbial CO2 production was stimulated within a larger zone around the roots (radius: c. 10 mm). This gave rise to a distinct pattern with a CO2 minimum at the root surface and a CO2 maximum c. 2 mm away from the root. In darkness, CO2 uptake ceased, and the CO2 -depleted zone disappeared within 2 h. By contrast, the oxygenated root zone remained even after 8 h, but diminished markedly over time. A tight coupling between photosynthetic processes and the spatiotemporal dynamics of O2 and CO2 in the rhizosphere of Lobelia was demonstrated, and we suggest that O2 -induced stimulation of the microbial community in the sediment increases the supply of inorganic carbon for photosynthesis by building up a CO2 reservoir in the rhizosphere.

A Method for Imaging Oxygen Distribution and Respiration at a Microscopic Level of Resolution.

Conventional oxygen (micro-) sensors assess oxygen concentration within a particular region or across a transect of tissue, but provide no information regarding its bidimensional distribution. Here, a novel imaging technology is presented, in which an optical sensor foil (i.e., the planar optode) is attached to the surface of the sample. The sensor converts a fluorescent signal into an oxygen value. Since each single image captures an entire area of the sample surface, the system is able to deduce the distribution of oxygen at a resolution level of few micrometers. It can be deployed to dynamically monitor oxygen consumption, thereby providing a detailed respiration map at close to cellular resolution. Here, we demonstrate the application of the imaging tool to developing plant seeds; the protocol is explained step by step and some potential pitfalls are discussed.

MultiSense: A Multimodal Sensor Tool Enabling the High-Throughput Analysis of Respiration.

The high-throughput analysis of respiratory activity has become an important component of many biological investigations. Here, a technological platform, denoted the "MultiSense tool," is described. The tool enables the parallel monitoring of respiration in 100 samples over an extended time period, by dynamically tracking the concentrations of oxygen (O2) and/or carbon dioxide (CO2) and/or pH within an airtight vial. Its flexible design supports the quantification of respiration based on either oxygen consumption or carbon dioxide release, thereby allowing for the determination of the physiologically significant respiratory quotient (the ratio between the quantities of CO2 released and the O2 consumed). It requires an LED light source to be mounted above the sample, together with a CCD camera system, adjusted to enable the capture of analyte-specific wavelengths, and fluorescent sensor spots inserted into the sample vial. Here, a demonstration is given of the use of the MultiSense tool to quantify respiration in imbibing plant seeds, for which an appropriate step-by-step protocol is provided. The technology can be easily adapted for a wide range of applications, including the monitoring of gas exchange in any kind of liquid culture system (algae, embryo and tissue culture, cell suspensions, microbial cultures).

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering.

Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen-imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell-seeding strategy. Immortalized hMSCs, pre-cultured in a monolayer for 30-40% or 70-80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real-time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle-type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30-40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70-80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell-seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two-dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894-902. © 2016 Wiley Periodicals, Inc.

Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence.

In Salmonella infection, the Salmonella pathogenicity island-2 (SPI-2)-encoded type three secretion system (T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin-pretreated mouse model SPI-2-dependent Salmonella replication in lamina propria CD11c(-)CXCR1(-) monocytic phagocytes/macrophages (MΦ) is required for the development of colitis. In addition, containment of intracellular Salmonella in the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase (PHOX), and possibly type 2 nitric oxide synthase (NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasive Salmonella infection in the gut tissue and its impact on Salmonella-MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model of Salmonella enterocolitis using luminescence two-dimensional in vivo oxygen imaging. We found that gut tissue oxygen levels dropped from ∼78 Torr (∼11% O2) to values of ∼16 Torr (∼2% O2) during infection. Because in vivo virulence of Salmonella depends on the Salmonella survival in MΦ, Salmonella-MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild-type and t3ss2 non-expressing Salmonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species (ROS) production and reduced Salmonella ROS perception. In addition, hypoxia enhanced SPI-2 transcription and translocation of SPI-2-encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia on Salmonella replication under normoxic conditions. However, if t3ss2 non-expressing Salmonella were used, hypoxia did not further enhance Salmonella recovery in a PHOX and NOS2-deficient situation. Hence, these data suggest that hypoxia-induced impairment of antimicrobial activity and Salmonella virulence cooperate to allow for enhanced Salmonella replication in MΦ.

Quantitative imaging of rhizosphere pH and CO2 dynamics with planar optodes.

BACKGROUND AND AIMS: Live imaging methods have become extremely important for the exploration of biological processes. In particular, non-invasive measurement techniques are key to unravelling organism-environment interactions in close-to-natural set-ups, e.g. in the highly heterogeneous and difficult-to-probe environment of plant roots: the rhizosphere. pH and CO2 concentration are the main drivers of rhizosphere processes. Being able to monitor these parameters at high spatio-temporal resolution is of utmost importance for relevant interpretation of the underlying processes, especially in the complex environment of non-sterile plant-soil systems. This study introduces the application of easy-to-use planar optode systems in different set-ups to quantify plant root-soil interactions. METHODS: pH- and recently developed CO2-sensors were applied to rhizobox systems to investigate roots with different functional traits, highlighting the potential of these tools. Continuous and highly resolved real-time measurements were made of the pH dynamics around Triticum turgidum durum (durum wheat) roots, Cicer arietinum (chickpea) roots and nodules, and CO2 dynamics in the rhizosphere of Viminaria juncea. KEY RESULTS: Wheat root tips acidified slightly, while their root hair zone alkalized their rhizosphere by more than 1 pH unit and the effect of irrigation on soil pH could be visualized as well. Chickpea roots and nodules acidified the surrounding soil during N2 fixation and showed diurnal changes in acidification activity. A growing root of V. juncea exhibited a large zone of influence (mm) on soil CO2 content and therefore on its biogeochemical surrounding, all contributing to the extreme complexity of the root-soil interactions. CONCLUSIONS: This technique provides a unique tool for future root research applications and overcomes limitations of previous systems by creating quantitative maps without, for example, interpolation and time delays between single data points.

Ratiometric luminescence 2D in vivo imaging and monitoring of mouse skin oxygenation.

Tissue oxygenation plays a critical role in the pathogenesis of various diseases, but non-invasive, robust and user-friendly methods for its measurement in vivo still need to be established. Here, we are presenting an in vivo oxygen-detection system that uses ratiometric luminescence imaging (RLI) as a readout scheme to determine the skin oxygen tension of mouse hind footpads via side-by-side comparison with more established techniques including luminescence-lifetime imaging using planar sensor films and the polarographic electrode as the gold standard. We also demonstrate that this technology allows the detection of changes in mouse skin tissue oxygenation induced by subjecting mice to systemic hypoxia. The data demonstrate oxygen imaging based on RLI to be a most useful tool for reliably and easily analyzing and monitoring skin tissue oxygenation in vivo. This technology will advance our understanding of local regulation of skin tissue oxygenation in various disease conditions.

An imaging method for oxygen distribution, respiration and photosynthesis at a microscopic level of resolution.

Biological samples are far from homogeneous, with complex compartmentation being the norm. Major physiological processes such as respiration do not therefore occur in a uniform manner within most tissues, and it is currently not possible to image its gradients in living plant tissues. A compact fluorescence ratiometric-based device is presented here, consisting of an oxygen-sensitive foil and a USB (universal serial bus) microscope. The sensor foil is placed on the sample surface and, based on the localized change in fluorescence signal over time, information about the oxygen consumption (respiration) or evolution (photosynthesis) can be obtained. Using this imaging technique, it was possible to demonstrate the spatial pattern of oxygen production and consumption at a c. 20-µm level of resolution, and their visualization in the rhizosphere, stem and leaf, and within the developing seed. The oxygen mapping highlighted the vascular tissues as the major stem sink for oxygen. In the leaf, the level of spatial resolution was sufficient to visualize the gas exchange in individual stomata. We conclude that the novel sensor set-up can visualize gradients in oxygen-consuming and producing processes, thereby facilitating the study of the spatial dynamics of respiration and photosynthesis in heterogeneous plant tissues.

Visualisation of cortical pO(2) during an epidural mass lesion in rodents.

Monitoring p(bt)O(2) is a valuable supplemental -procedure for neurocritically ill patients. Here, we utilise an opto-chemical method for measuring cortical pO(2) during a reversibly introduced epidural mass lesion in a rat model. The sensor was placed in a cortical window of 17 ventilated Wistar rats. Inflating the balloon device over the contralateral hemisphere increased ICP. Physiological parameters and cortical pO(2) were recorded. The ICP increased from 6.2 ± 4.6 to 44.6 ± 12.6 mmHg (p < 0.001). Cortical pO(2) over arterioles changed from 28.9 ± 2.1 to 19.0 ± 2.1 mmHg (p < 0.001), over venules from 14.8 ± 1.2 to 9.9 ± 1.5 mmHg (p = 0.002) and over parenchyma from 4.1 ± 0.7 to 2.4 ± 0.8 mmHg respectively (p < 0.001), while basic physiological parameters remained constant (p > 0.05). During baseline, arterial pO(2) correlated significantly with cortex arteriole and venole pO(2), but not with cortex parenchyma pO(2). While ICP was raised, cortical pO(2) did not correlate with arterial pO(2). In this model, a moderate epidural mass lesion causes a significant decrease in cortical pO(2). Cortex parenchyma pO(2) appeared to be independent from arterial pO(2). The correlation of cortex vessel pO(2) with arterial pO(2) disappeared during the epidural mass lesion. These findings show the capability of the device to elucidate the behaviour of functionally different cortex areas at pathophysiological conditions.

Radial oxygen gradients over rat cortex arterioles.

PURPOSE: We present the results of the visualisation of radial oxygen gradients in rats' cortices and their potential use in neurocritical management. METHODS: PO2 maps of the cortex of ten sedated, intubated and controlled ventilated Wistar rats were obtained with a camera (SensiMOD, PCO, Kelheim, Germany). Those pictures were analysed and edited by a custom-made software. A virtual matrix, designed to evaluate the cortical O2 partial pressure, was placed vertically to the artery under investigation, and afterwards multiple regions of interest were measured (width 10 pixels, length 15-50 pixels). The results showed a map of the cerebral oxygenation, which allowed us to calculate radial oxygen gradients over arterioles. Three groups were defined according to the level of the arterial pO2: PaO2 < 80, PaO2 80-120 and PaO2 > 120. Gradients were analysed from the middle of the vessel to its border (1), from the border into the parenchyma next to the vessel (2) and a combination of both (3). RESULTS: Gradient 1 showed significantly different cortical pO2 values between the three different groups. The mean pO2 values were 2.62, 5.29 and 5.82 mmHg/mm. Gradient 2 measured 0.56, 0.90 and 1.02 mmHg/mm respectively. Gradient 3 showed significant results between the groups with values of 3.18, 6.19 and 6.84 mmHg/mm. CONCLUSION: Using these gradients, it is possible to describe and compare the distribution of oxygen to the brain parenchyma. With the presented technique, it is possible to detect pO2 changes in the oxygen supply of the brain cortex.

Simultaneous imaging of cortical partial oxygen pressure and anatomic structures using a transparent optical sensor foil.

BACKGROUND/PURPOSE: Reliable information of cerebral oxygenation is-besides the monitoring of the intracranial pressure-of eminent interest when treating patients with brain injuries. In this study, we introduce a new, fast, and sensitive method capable of determining the cortical partial oxygen pressure on the surface of the cortex using a special sensor foil. METHODS: The introduced method exploits the O2-dependent phosphorescence of a thin sensor foil, which is excited by a short light-emitting diode flash. The optical signal is registered by a charge-coupled device camera and analyzed with PC-based software. The adequacy of this method was tested in 10 animals. The sensor device was placed directly over the cortex after craniotomy and removal of the dura. Arterial oxygen pressure was systematically varied by modifying the ventilation gas mixture. A total of 225 measurements were performed within 4 regions of interest. RESULTS: Obtained results were sufficient in each case. The pO2 over the cortex correlated well with arterial pO2. Measurements over arteries showed a correlation coefficient of 0.72 (P<0.001), over veins 0.58 (P<0.001), over cortical parenchyma 0.46 (P<0.001), and in a larger region of interest containing vessels and cortical tissue 0.59 (P<0.001). The frequency of the measurements was 7 Hz with a single measurement covering an area of 30 x 30 microm. CONCLUSIONS: For the first time, nearly online pO2 maps of a brain cortex can be generated, allowing simultaneously also separate measurements over distinct anatomic structures yielding a good spatial resolution.