Local PO2 Research Articles

Correlation between local instantaneous dose rate and oxygen pressure reduction during proton pencil beam scanning irradiation

Background and purposeOxygen dynamics may be important for the tissue-sparing effect observed at ultra-high dose rates (FLASH sparing effect). This study investigated the correlation between local instantaneous dose rate and radiation-induced oxygen pressure reduction during proton pencil beam scanning (PBS) irradiations of a sample and quantified the oxygen consumption g-value. Materials and methodsA 0.2 ml phosphorescent sample (1 μM PtG4 Oxyphor probe in saline) was irradiated with a 244 MeV proton PBS beam. Four irradiations were performed with variations of a PBS spot pattern with 5 × 7 spots. During irradiation, the partial oxygen pressure (pO2) was measured with 4.5 Hz temporal resolution with a phosphorometer (Oxyled) that optically excited the probe and recorded the subsequently emitted light. A calibration was performed to calculate the pO2 level from the measured phosphorescence lifetime. A fiber-coupled scintillator simultaneously measured the instantaneous dose rate in the sample with 50 kHz sampling rate. The oxygen consumption g-value was determined on a spot-by-spot level and using the total pO2 change for full spot pattern irradiation. ResultsA high correlation was found between the local instantaneous dose rate and pO2 reduction rate, with a correlation coefficient of 0.96–0.99. The g-vales were 0.18 ± 0.01 mmHg/Gy on a spot-by-spot level and 0.17 ± 0.01 mmHg/Gy for full spot pattern irradiation. ConclusionsThe pO2 reduction rate was directly related to the local instantaneous dose rate per delivered spot in PBS deliveries. The methodology presented here can be applied to irradiation at ultra-high dose rates with modifications in the experimental setup.

Passive and active oxidation of Al in the Al–Al2O3 composite refractory at high temperatures in air

The active and passive oxidation of Al in Al–Al2O3 composites was investigated in air at high temperatures. As fresh air impinged on the surface of the Al–Al2O3 composite sample, a thin Al2O3 layer was preferentially formed via passive oxidation of Al, which consumed most of the oxygen and lowered the local Po2 inside the sample. At low Po2, active oxidation of Al occurred and predominated in the interior transition layer, which further lowered the local Po2 to extremely low values within the non-oxide stability field. The local equilibrium PAlxOy generated by the active oxidation of Al reached a micro-positive pressure state and served as a gas barrier, hindering the inward diffusion of O2. The Po2 inside the composite was lowered by Al in the outer layers via a self-gettering process. Subsequently, Al4O4C and Al2OC-AlN solid solution were formed by both the direct reaction of Al(l) and the indirect reaction of AlxOy(g). After sintering in air, the Al–Al2O3 composite exhibited a functional gradient structure with a thin oxide layer and an interior non-oxide-reinforced corundum. A reaction mechanism model was established.

Dynamics of PO2 and VO2 in resting and contracting rat spinotrapezius muscle.

This study examined changes in interstitial PO2, which allowed calculation of VO2 during periods of rest, muscle contraction and recovery using an in situ rat spinotrapezius muscle preparation. The PO2 was measured using phosphorescence quenching microscopy and the muscle VO2 was calculated as the rate of O2 disappearance during brief periods of muscle compression to stop blood flow with a supra-systolic pressure. The PO2 and VO2 measurements were made during "5s compression and 15s recovery" (CR) cycles. With all three stimulation frequencies, 1, 2 and 4Hz, the fall in interstitial PO2 and rise in VO2 from resting values occurred within the first 20s of contraction. The PO2 during contraction became lower as stimulation frequency increased from 1 to 4Hz. VO2 was higher at 2Hz than at 1Hz contraction. With cessation of stimulation, PO2 began increasing exponentially towards baseline values. After 1 and 2Hz contraction, the fall in muscle VO2 was delayed by one CR cycle and then exponentially decreased towards resting values. After 4Hz stimulation, VO2 increased for 2 cycles and then decreased. The post-contraction transients of PO2 and VO2 were not synchronous and had different time constants. With further analysis two distinct functional responses were identified across all stimulation frequencies having PO2 during contraction above or below 30mmHg. The corresponding VO2 responses were different - for "high" PO2, muscle VO2 reached high levels, while for the "low" PO2 data set muscle VO2 remained low. Recovery patterns were similar to those described above. In summary, local microscopic PO2 and VO2 were measured in resting and contracting muscle in situ and the post-contraction transients of PO2 and VO2 were all much slower than the onset transients.

Oxygen therapeutic window induced by myo-inositol trispyrophosphate (ITPP)-Local pO2 study in murine tumors.

Hypoxia, an inevitable feature of locally advanced solid tumors, has been known as an adverse prognostic factor, a driver of an aggressive phenotype, and an unfavorable factor in therapies. Myo-inositol trispyrophosphate (ITPP) is a hemoglobin modifier known to both increase O2 release and normalize microvasculature. Our goal was to measure the tumor oxygen partial pressure dynamic changes and timing of the therapeutic window after ITPP systemic administration. Two syngeneic tumor models in mice, B16 melanoma and 4T1 breast carcinoma, were used, with varying ITPP dose schedules. Tissue oxygenation level was measured over several days in situ in live animals by Electron Paramagnetic Resonance oximetry with implanted OxyChip used as a constant sensor of the local pO2 value. Both B16 and 4T1 tumors became more normoxic after ITPP treatment, with pO2 levels elevated by 10-20 mm Hg compared to the control. The increase in pO2 was either transient or sustained, and the underlying mechanism relied on shifting hypoxic tumor areas to normoxia. The effect depended on ITPP delivery intervals regarding the tumor type and growth rate. Moreover, hypoxic tumors before treatment responded better than normoxic ones. In conclusion, the ITPP-generated oxygen therapeutic window may be valuable for anti-tumor therapies requiring oxygen, such as radio-, photo- or immunotherapy. Furthermore, such a combinatory treatment can be especially beneficial for hypoxic tumors.

Skeletal muscle blood flow response to local PO2 is impaired in high fat diet fed Sprague Dawley and Zucker Diabetic Sprague Dawley rats

Objective: Quantify the capillary blood flow responses to dynamic changes in oxygen concentrations ([O2]) in 27-week-old Sprague Dawley (SD) and Zucker Diabetic Sprague Dawley (ZDSD) type 2 diabetic (T2D) rats during hyperinsulinemic-euglycemic clamp. Hypothesis: Chronic hyperglycemia and elevated insulin in T2D causes impaired oxygen mediated blood flow regulation leading to functional defects in capillary network blood flow distribution at rest, during hyperinsulinemia, and in response to oxygen challenges. Methods: 8 SD and 10 ZDSD rats were fed high fat (HF) diet upon arrival at 15 weeks old, transitioned to a HF and high sugar diet from 16-19 weeks old, to induce T2D in the ZDSD strain, before being returned to HF until intravital video microscopy (IVVM). At 27 weeks of age, animals were fasted overnight and on the morning of the experiment animals were anaesthetized, mechanically ventilated, and catheters were introduced for systemic monitoring, fluid resuscitation and anaesthetic administration. The extensor digitorum longus muscle was isolated, blunt dissected, and reflected over a microfluidic gas exchange chamber fitted in the stage of an inverted microscope. Microvascular blood flow responses to sequential changes in [O2] (7%-12%-2%-7%) were recorded at baseline and during hyperinsulinemic-euglycemic clamp. Hyperinsulinemic-euglycemic clamp was achieved by simultaneous infusion of insulin (2U/hr/kg) and variable rate of 50% glucose until euglycemia was reached (5-7 mM). ZDSD rats with fasting blood glucose <20 mM on the day of IVVM were excluded from the hyperglycemic group. Analysis of IVVM videos was completed offline using custom MATLAB software. Animal protocols were approved by Memorial University’s Animal Care Committee. Results: SD red blood cell (RBC) saturation (SO2) significantly increased in response to 12% [O2] at both baseline and clamp (62.0 ± 5.9 vs. 75.8 ± 3.8 %, p <0.0001, and 62.7 ± 8.2 vs. 76.7 ± 6.5 %, p = 0.0001), RBC SO2 significantly decreased with 2% [O2] under both conditions compared to 7% (62.0 ± 5.9 vs. 38.1 ± 6.3 %, p <0.0001, and 62.7 ± 8.2 vs. 46.0 ± 9.5 %, p <0.0001). RBC velocity, supply rate (SR) and hematocrit showed no change from 7% (174.8 ± 59.7 μm/s, 10.7 ± 3.4 cells/s, and 27.5 ± 3.8 %) in response to increased or decreased [O2]. ZDSD rats had a significant increase between baseline and clamp in RBC SR at 12% (19.5 ± 3.3 vs 23.7 ± 9.1 cells/s, p = 0.0151) as well as at 2% (14.9 ± 2.6 vs 23.9 ± 8.0 cells/s, p = 0.0295). However, there was no change in RBC velocity or hematocrit in the ZDSD strain in response to imposed [O2] changes at baseline and clamp. Conclusion: SD and ZDSD animals lacked expected responses in capillary RBC velocity, SR, and hematocrit to imposed local [O2] changes both at baseline and during euglycemic clamp. This finding suggests that both hyperglycemia in T2D and high fat feeding alone affects microvascular blood flow responses to acute changes in local oxygen concentrations. This project was supported by the Canadian Institute of Health Research through a project grant to GMF. Studentships were supported by Memorial University's School of Graduate Studies and the Faculty of Medicine. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chemical Looping Combustion of a Biomass Char in Fe2O3-, CuO-, and SrFeO3−δ-Based Oxygen Carriers

This research focuses on the combustion of biomass char in fluidized beds of various particulate solids, which, under the conditions of the reaction, were either inert or capable of supplying oxygen to reactions. The latter were termed oxygen carriers. The solids used were SiO2, as an inert material, and three oxygen carriers: (1) Fe2O3 prepared from a natural pyrite ore, (2) CuO supported on mayenite, and (3) SrFeO3−δ strontium ferrite perovskite. Combustion experiments were undertaken by introducing a sample of partially devolatilized biomass (commercial “biochar”) to a hot bubbling bed (inner diameter of 30 mm), fluidized by a mixture of oxygen and nitrogen, then analyzing the composition of the off-gas and the burnout time of the char sample. In the temperature range investigated in this work (1023–1168 K), CuO and SrFeO3−δ but not Fe2O3 thermally decomposed, releasing gaseous O2 [so-called “chemical looping oxygen uncoupling” (CLOU)]. Hence, to make the combustion conditions comparable to various oxygen carriers, all experiments were performed using a fluidizing gas with a fixed partial pressure of O2 (pO2) of ∼0.015 bar. Despite the same nominal pO2, the occurrence of the oxygen uncoupling reaction increased the total net amount of O2(g) available in the process, affecting external mass transfer of O2 to the char particle and accelerating its rate of combustion. The time needed to totally combust 0.1 g of biochar particles in different beds at 1168 K followed the trend CuO < SrFeO3−δ < Fe2O3 ≈ silica sand. The difference in the performance of CuO and SrFeO3−δ was ascribed to the lower oxygen availability via CLOU in perovskite compared to copper oxide. Interestingly, combustion in the bed of Fe2O3 particles took a similar amount of time as combustion in the inert bed of SiO2, despite iron oxide playing an active role in the process. The finding is explained by Fe2O3 reacting with CO produced from incomplete char combustion, which results in the reduced oxide competing with char for O2(g) and effectively decreasing the local pO2.

PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study

PurposePEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan, an important component of the extracellular matrix, increasing the delivery of therapeutic molecules. Combinations of chemotherapy and PEGPH20, however, have been unsuccessful in Phase III clinical trials. We hypothesize that by increasing tumor oxygenation by improving vascular patency and perfusion, PEGPH20 will also act as a radiosensitization agent. Experimental DesignThe effect of PEGPH20 on radiation treatment was analyzed with respect to tumor growth, survival time, p02, local blood volume, and the perfusion/permeability of blood vessels in a human pancreatic adenocarcinoma BxPC3 mouse model overexpressing hyaluronan synthase 3 (HAS3). ResultsMice overexpressing HAS3 developed fast growing, radiation resistant tumors that became rapidly more hypoxic as time progressed. Treatment with PEGPH20 increased survival times when used in combination with radiation therapy, significantly more than either radiation therapy or PEGPH20 alone. In mice that overexpressed HAS3, EPR imaging showed an increase in local pO2 that could be linked to increases in perfusion/permeability and local blood volume immediately after PEGPH20 treatment. Hyperpolarized [1-13C] pyruvate suggested PEGPH20 caused a metabolic shift towards decreased glycolytic flux. These effects were confined to the mice overexpressing HAS3 - no effect of PEGPH20 on survival, radiation treatment, or pO2 was seen in wild type BxPC3 tumors. ConclusionsPEGPH20 may be useful for radiosensitization of pancreatic cancer but only in the subset of tumors with substantial hyaluronan accumulation. The response of the treatment may potentially be monitored by non-invasive imaging of the hemodynamic and metabolic changes in the tumor microenvironment.

Theoretical Analysis of Critical Conditions for Crack Formation and Propagation, and Optimal Operation of SOECs

A theoretical analysis on crack formation and propagation was performed based on the coupling between the electrochemical process, classical elasticity, and fracture mechanics. The chemical potential of oxygen, thus oxygen partial pressure, at the oxygen electrode-electrolyte interface () was investigated as a function of transport properties, electrolyte thickness and operating conditions (e.g., steam concentration, constant current, and constant voltage). Our analysis shows that: a lower ionic area specific resistance (ASR), and a higher electronic ASR () of the oxygen electrode/electrolyte interface are in favor of suppressing crack formation. The thus local pO2, are sensitive towards the operating parameters under galvanostatic or potentiostatic electrolysis. Constant current density electrolysis provides better robustness, especially at a high current density with a high steam content. While constant voltage electrolysis leads to greater variations of Constant current electrolysis, however, is not suitable for an unstable oxygen electrode because can reach a very high value with a gradually increased A crack may only occur under certain conditions when

Design concept of co-ionic conducting solid oxide electrolyte for stable operation in a cell-imbalanced fuel cell stack

A bilayer composite electrolyte comprising BaCe0.85Y0.15O3-d (BCY15) – Gd0.2Ce0.8O2-d (GDC20) at the anode side and BaZr0.85Y0.15O3-d (BZY15) – Nd0.1Ce0.9O2-d (NDC10) at the cathode side is designed for improving the stability of co-ionic conducting solid oxide fuel cells (SOFCs) in a cell imbalanced stack. Contrary to the single layer structured SOFCs, the bi-layer cell stably operates without electrode delamination under negative voltage conditions. We measure local internal pO2 values using embedded Pt probes. Based on these values, local electronic conduction is estimated in combination with four probe DC conductivity measurements. It is found that n-type conductivity (∼10−3 Scm−1) and p-type conductivity (10−5–10−4 Scm−1) are developed in the BCY15-GDC20 near the anode side and in the BZY15-NDC10 near the cathode side, respectively. The results indicate that local electronic conduction in electrolyte regions near both the anode and cathode interfaces is a crucial factor for the durability of co-ionic SOFCs under negative voltage operation. We therefore suggest the bi-layer configuration as a practical solution to protect co-ionic SOFCs in a cell-imbalanced stack.

Effect of integrated technology for fattening beef cattle in the livestock zone of North Sulawesi

This on-farm study aims to increase beef cattle productivity by utilizing integrated technology packages for local cattle fattening with a target of at least 800g/head daily gain. This study was carried out in the Minahasa Selatan district from August to October 2018. The integrated technology package for the fattening business pattern included: (1) complete feed, (2) intensive maintenance in multi-purpose cages, and (3) eradication of complete parasites. A complete feed is formulated in a balanced feed consisting of ammoniated rice straw and concentrate locally. The amount of dry matter concentrate was given per head is 1.5% of body weight (50% total consumption), while the ammoniated straw was ad libitum. For multipurpose cages, the upper part is equipped with a multipurpose tower as a barn while simultaneously serving straw, ad libitum, and continuous. Complete eradication of parasites using broad-spectrum anti-parasites to eradicate endoparasites that attack digestive tissues and other internal organs and ectoparasites to control the skin worms and eyeworms, cattle grubs, mange mites, sucking lice, sand tampans, and ticks. This study used fifteen local PO bulls fattened with an initial body weight of an average of 278.4 kg (± 54.34) with an observation period of 12 weeks. The observations showed that the average daily gain was 1,158 g/head, feed consumption 8,358 kg DM/head/day, and feed conversion ratio of 7.56. In contrast, the results of the business feasibility analysis showed a BCR coefficient of 1.69. The results of statistical analysis showed that the average weight gain increased significantly (p <0.01) after utilizing integrated fattening technology (308.8vs278.4 kg). It was concluded that the use of integrated technology for cattle fattening in beef cattle could increase productivity significantly and economically feasible.

The Relative Superiority of Crossbred Using Friesian Holstein Hybrid Cement Bull to Local Ongole Hybrid Cattle

The purpose of this study was to analyze the relative superiority of crossbred to local Ongole hybrid (PO) cattle. This research was carried out for 18 months in the Konawe Selatan, and Kolaka Timur Regency, Southeast Sulawesi Province. The number of cows used was 48 cows, and the cement used was Friesian Holstein Hybrid (PFH) Cement and Ongole hybrid (PO) Cows from BBIB Lembang. Data were analyzed by the general linear model (General Liner Model) with the source of diversity was genotype and sex of calf. Based on the results of the study concluded that the crossbred calf has a relatively high relative advantage over local Ongole hybrid cattle with an average value of 7.76-11.28%. The crossing of Friesian Holstein Hybrid (PFH) cows with Ongole hybrid (PO) parents resulted in offspring with PFPO genotype with an average relative superiority value (for all parameters) of 11.28%, higher than the PFS genotype of 7.65% and PFL of 9.60%. The Result of this crossing increases meat production, and it is recommended to crossbreed PFH cows with PO mothers; however, they still consider their suitability to the local environment and the purity of local PO cows.

Regional pulmonary perfusion patterns in humans are not significantly altered by inspiratory hypercapnia.

Pulmonary vascular tone is known to be sensitive to both local alveolar Po2 and Pco2. Although the effects of hypoxia are well studied, the hypercapnic response is relatively less understood. We assessed changes in regional pulmonary blood flow in humans in response to hypercapnia using previously developed MRI techniques. Dynamic measures of blood flow were made in a single slice of the right lung of seven healthy volunteers following a block-stimulus paradigm (baseline, challenge, recovery), with CO2 added to inspired gas during the challenge block to effect a 7-Torr increase in end-tidal CO2. Effects of hypercapnia on blood flow were evaluated based on changes in spatiotemporal variability (fluctuation dispersion, FD) and in regional perfusion patterns in comparison to hypoxic effects previously studied. Hypercapnia increased FD 2.5% from baseline (relative to control), which was not statistically significant (P = 0.07). Regional perfusion patterns were not significantly changed as a result of increased (P = 0.90). Reanalysis of previously collected data using a similar protocol but with the physiological challenge replaced by decreased ( = 0.125) showed marked flow redistribution (P = 0.01) with the suggestion of a gravitational pattern, demonstrating hypoxia has the ability to affect regional change with a global stimulus. Taken together, these data indicate that hypercapnia of this magnitude does not lead to appreciable changes in the distribution of pulmonary perfusion, and that this may represent an interesting distinction between the hypoxic and hypercapnic regulatory response.NEW & NOTEWORTHY Although it is well known that the pulmonary circulation responds to local alveolar hypoxia, and that this mechanism may facilitate ventilation-perfusion matching, the relative role of CO2 is not well appreciated. This study demonstrates that an inspiratory hypercapnic stimulus is significantly less effective at inducing changes in pulmonary perfusion patterns than inspiratory hypoxia, suggesting that in these circumstances hypercapnia is not sufficient to induce substantial integrated feedback control of ventilation-perfusion mismatch across the lung.

Automated Evaluation of Microvascular Blood Flow and Oxygenation following Trauma and Hemorrhage in Rats

Studies evaluating blood flow and oxygen partial pressure (PO2) may not directly measure both parameters and may depend on investigator's selection of measuring sites. In addition, relevant systemic parameters may not be simultaneously recorded. Therefore, we implemented a new automated system for blood flow and PO2 acquisition in large tissue areas while collecting systemic information. In 10 animals anesthetized with isoflurane, cardio‐respiratory parameters were continuously recorded, in experiments lasting up to 4 h. Other data were collected at baseline, and after laparotomy and 30 min hemorrhage (40% of total blood volume). A cremaster muscle was spread over a thermostatically‐controlled pedestal fixed to a motorized stage. Sixteen noninvasive PO2 measurements using oxygen‐dependent phosphorescence quenching and fiber‐optics were performed during a computer‐controlled tissue scan. In the same areas used for PO2 measurements, microvascular blood flow was estimated employing laser speckle contrast imaging. Blood was sampled for extensive biochemistry and coagulation profiles. The system was used successfully by different operators. One set of flow and PO2 measurements was completed in less than 90 s. Changes in muscle flow correlated with local PO2 but several systemic parameters did not correlate with blood flow and PO2, emphasizing the importance of performing both local and systemic evaluations. System advantages include integration between local and multiple systemic parameters, unbiased data collection/analysis, easy implementation, improved performance, no need for customized programming, and simplified training compared to intravital microscopy.Support or Funding InformationSupported by US Army Medical Research and Materiel Command.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of shifting epithelial Na+ transport on renal medullary oxygen tension: Modeling and analysis

The mammalian kidney is particularly vulnerable to hypoperfusion, because O2 supply to the renal medulla barely exceeds its O2 requirements. Renal O2 consumption is mainly driven by the metabolic work of tubular Na+ reabsorption. In this study, we examined the impact of shifting the Na+ transport site on medullary oxygen tension (PO2) of the rat kidney. To accomplish that goal, we extended our published model of renal epithelial transport to include renal vasculature and to simulate the crosstalk among nephron segments and vasculature. The model represents the complex structural organization of the medulla, which is believed to have a significant impact on medullary O2 distribution. The transport of red blood cells, hemoglobin, and O2 was explicitly represented. We considered basal cellular O2 consumption and O2 consumption for Na+/K+‐ATPase‐mediated transport. Our model predicts that the structural organization of the outer medulla results in significant Po2 gradients in the axial and radial directions. The segregation of descending vasa recta, the main supply of O2, at the center and immediate periphery of the vascular bundles gives rise to large radial differences in Po2, limits O2 reabsorption from long descending vasa recta, and helps preserve O2 delivery to the inner medulla. When Na+ delivery to the S3 and thick ascending limb segments is elevated (e.g., by pharmacological manipulation), local interstitial PO2 may decrease significantly, a result that indicates elevated risk of hypoxia.Support or Funding InformationThis research was supported by the Canada 150 Research Chair program and by the National Institutes of Health: National Institute of Diabetes and Digestive and Kidney Diseases, grant R01DK106102.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Merger of dynamic two-photon and phosphorescence lifetime microscopy reveals dependence of lymphocyte motility on oxygen in solid and hematological tumors

BackgroundLow availability of oxygen in tumors contributes to the hostility of the tumor microenvironment toward the immune system. However, the dynamic relationship between local oxygen levels and the immune surveillance of tumors by tumor infiltrating T-lymphocytes (TIL) remains unclear. This situation reflects a methodological difficulty in visualizing oxygen gradients in living tissue in a manner that is suitable for spatiotemporal quantification and contextual correlation with individual cell dynamics tracked by typical fluorescence reporter systems.MethodsHere, we devise a regimen for intravital oxygen and cell dynamics co-imaging, termed ‘Fast’ Scanning Two-photon Phosphorescence Lifetime Imaging Microscopy (FaST-PLIM). Using FaST-PLIM, we image the cellular motility of T-lymphocytes in relation to the microscopic distribution of oxygen in mouse models of hematological and solid tumors, namely in bone marrow with or without B-cell acute lymphocytic leukemia (ALL), and in lungs with sarcoma tumors.ResultsBoth in bone marrow leukemia and solid tumor models, TILs encountered regions of varying oxygen concentrations, including regions of hypoxia (defined as pO2 below 5 mmHg), especially in advanced-stage ALL and within solid tumor cores. T cell motility was sustained and weakly correlated with local pO2 above 5 mmHg but it was very slow in pO2 below this level. In solid tumors, this relationship was reflected in slow migration of TIL in tumor cores compared to that in tumor margins. Remarkably, breathing 100% oxygen alleviated tumor core hypoxia and rapidly invigorated the motility of otherwise stalled tumor core TILs.ConclusionsThis study demonstrates a versatile and highly contextual FaST-PLIM method for phosphorescence lifetime-based oxygen imaging in living animal tumor immunology models. The initial results of this method application to ALL and solid lung tumor models highlight the importance of oxygen supply for the maintenance of intratumoral T cell migration, define a 5 mmHg local oxygen concentration threshold for TIL motility, and demonstrate efficacy of supplementary oxygen breathing in TIL motility enhancement coincident with reduction of tumor hypoxia.

Fluorescence Lifetime Spectroscopy of the Cornea using a Multichannel Scaler

An interface based on Multichannel scaler (MCS; SR430; Stanford Research Systems, USA) has been tested for enabling fluorescence lifetime measurements of pO2-sensitive fluorophores with a custom-made confocal scanning microfluorometer (CSMF) for transcorneal pO2 measurements. SR430 served as a transient photon counter. A trigger pulse, coincident with the rising edge of an excitation pulse for fluorescence, was coupled to accumulate emission pulses detected by a photomultiplier tube (PMT; R928 HA, Hamamatsu, Inc). The pulse started the photon counting and segmented the photon count data into consecutive time bins (ranging from 1 up to 32k), the width of which could be varied from 5 ns to 10.486 ms. With no dead time between bins, photon counting with the fluorescence decay is seamless. Silica-bound Ruthenium phenanthroline, dispersed in silicone rubber, was employed as a fluorophore sensitive to pO2. The test interface for CSMF-MCS system showed sensitive pO2 measurements and hence is suitable to measure local pO2 at any depth across the cornea.

Real-time local oxygen measurements for high resolution cellular imaging

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O2 (pO2) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O2-micro-sensor created by adsorbing O2-sensitive and O2-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O2, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO2 and micro-gradients of pO2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO2 and intracellular events can be imaged simultaneously. In addition, the response-time (t0.5 = 0.7 s, 0–160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨm); and (2) OxySplots measuring pO2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca2+]i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

Age-Dependent Impairment of Neurovascular and Neurometabolic Coupling in the Hippocampus.

Neurovascular and neurometabolic coupling are critical and complex processes underlying brain function. Perturbations in the regulation of these processes are, likely, early dysfunctional alterations in pathological brain aging and age-related neurodegeneration. Evidences support the role of nitric oxide (•NO) as a key messenger both in neurovascular coupling, by signaling from neurons to blood vessels, and in neurometabolic coupling, by modulating O2 utilization by mitochondria. In the present study, we investigated the functionality of neurovascular and neurometabolic coupling in connection to •NO signaling and in association to cognitive performance during aging. For this, we performed in vivo simultaneous measurements of •NO, O2 and cerebral blood flow (CBF) in the hippocampus of F344 rats along chronological age in response to glutamatergic activation and in correlation with cognitive performance. Firstly, it is evidenced the temporal sequence of events upon glutamate stimulation of hippocampal dentate gyrus, encompassing the local and transitory increase of •NO followed by transitory local changes of CBF and pO2. Specifically, the transient increase of •NO is followed by an increase of CBF and biphasic changes of the local pO2. We observed that, although the glutamate-induced •NO dynamics were not significantly affected by aging, the correspondent hemodynamic was progressively diminished accompanying a decline in learning and memory. Noteworthy, in spite of a compromised blood supply, in aged rats we observed an increased ΔpO2 associated to the hemodynamic response, suggestive of a decrease in the global metabolic rate of O2. Furthermore, the impairment in the neurovascular coupling observed along aging in F344 rats was mimicked in young rats by promoting an unbalance in redox status toward oxidation via intracellular generation of superoxide radical. This observation strengthens the idea that oxidative stress may have a critical role in the neurovascular uncoupling underlying brain aging and dysfunction. Overall, data supports an impairment of neurovascular response in connection with cognition decline due to oxidative environment-dependent compromised •NO signaling from neurons to vessels during aging.

Combined in Vivo Amperometric Oximetry and Electrophysiology in a Single Sensor: A Tool for Epilepsy Research.

Seizures are paroxysmal events in which increased neuronal activity is accompanied by an increase in localized energetic demand. The ability to simultaneously record electrical and chemical events using a single sensor poses a promising approach to identify seizure onset zones in the brain. In the present work, we used ceramic-based platinum microelectrode arrays (MEAs) to perform high-frequency amperometric recording of local pO2 and local field potential (LFP)-related currents during seizures in the hippocampus of chronically implanted freely moving rats. Resting levels of O2 in the rodent brain varied between 6.6 ± 0.7 μM in the dentate gyrus (DG) region of the hippocampus and 22.1 ± 4.9 μM in the cerebral cortex. We also observed an expected increase in hippocampal pO2 (15 ± 4% from baseline) in response to tail pinch stress paradigm. Finally, induction of status epilepticus by intrahippocampal injection of pilocarpine induced biphasic changes in pO2 in the hippocampus. The initial dip at seizure onset (ΔO2 = -4.5 ± 0.7 μM) was followed by a prolonged hyperoxygenation phase (ΔO2 = +10.4 ± 2.9 μM). By acquiring the amperometry signal with a high sampling rate of 100 Hz we decomposed the raw signal in an oximetry recording (<1 Hz) and LFP recording (>1 Hz), demonstrating that each individual Pt site can simultaneously report changes in local pO2 and LFP-related currents during pilocarpine-induced seizure activity. This has high potential for translation into the clinical setting supported on intracranial grid or strip electrodes.

Abnormal oxygen homeostasis in the nucleus tractus solitarii of the spontaneously hypertensive rat

New Findings What is the central question of this study? Arterial hypertension is associated with impaired neurovascular coupling in the somatosensory cortex. Abnormalities in activity‐dependent oxygen consumption in brainstem regions involved in the control of cardiovascular reflexes have not been explored previously. What is the main finding and its importance? Using fast‐cyclic voltammetry, we found that changes in local tissue PO2 in the nucleus tractus solitarii induced by electrical stimulation of the vagus nerve are significantly impaired in spontaneously hypertensive rats. This is consistent with previous observations showing that brainstem hypoxia plays an important role in the pathogenesis of arterial hypertension. The effects of arterial hypertension on cerebral blood flow remain poorly understood. Haemodynamic responses within the somatosensory cortex have been shown to be impaired in the spontaneously hypertensive rat (SHR) model. However, it is unknown whether arterial hypertension affects oxygen homeostasis in vital brainstem areas that control cardiovascular reflexes. In this study, we assessed vagus nerve stimulation‐induced changes in local tissue PO2 (PtO2) in the caudal nucleus tractus solitarii (cNTS) of SHRs and normotensive Wistar rats. Measurements of PtO2 were performed using a novel application of fast‐cyclic voltammetry, which allows higher temporal resolution of O2 changes than traditional optical fluorescence techniques. Electrical stimulation of the central cut end of the vagus nerve (ESVN) caused profound reductions in arterial blood pressure along with biphasic changes in PtO2 in the cNTS, characterized by a rapid decrease in PtO2 (‘initial dip’) followed by a post‐stimulus overshoot above baseline. The initial dip was found to be significantly smaller in SHRs compared with normotensive Wistar rats even after ganglionic blockade. The post‐ESVN overshoot was similar in both groups but was reduced in Wistar rats after ganglionic blockade. In conclusion, neural activity‐dependent changes in tissue oxygen in brainstem cardiovascular autonomic centres are significantly impaired in animals with arterial hypertension.