Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of >13 μm/s and a partial pressure of O2 (pO2) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7−/− naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Gαi-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO2, tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.

Tumor hypoxia is a persistent obstacle for traditional therapies in solid tumors. Strategies for mitigating the effects of hypoxic tumor cells have been developed under the assumption that chronically hypoxic tumor cells were the central cause of treatment resistance. In this study, we show that instabilities in tumor oxygenation are a prevalent characteristic of three tumor lines and previous characterization of tumor hypoxia as being primarily diffusion-limited does not accurately portray the tumor microenvironment. Phosphorescence lifetime imaging was used to measure fluctuations in vascular pO 2 in rat fibrosarcomas, 9L gliomas, and R3230 mammary adenocarcinomas grown in dorsal skin-fold window chambers (n = 6 for each tumor type) and imaged every 2.5 minutes for a duration of 60 to 90 minutes. O 2 delivery to tumors is constantly changing in all tumors, resulting in continuous reoxygenation events throughout the tumor. Vascular pO 2 maps show significant spatial heterogeneity at each time point, as well as between time points. The fluctuations in oxygenation occur with a common periodicity within and between tumors, suggesting a common mechanism, but have tumor type-dependent spatial patterns. The widespread presence of fluctuations in tumor oxygenation has broad ranging implications for tumor progression, stress response, and signal transduction, which are altered by oxygenation/reoxygenation events. [Cancer Res 2008;68(14):5812-9]

The primary purpose of this study was to examine the kinetics of partial pressure of oxygen (pO 2 ) fluctuations in fibrosarcoma (FSA) and 9L tumors under air and O 2 breathing conditions. The overall hypothesis was that key factors relating to oxygen tension fluctuations would vary between the two tumor types and as a function of the oxygen content of the breathing gas. To assist in the interpretation of the temporal data, spatial pO 2 distributions were measured in 10 FSA and 8 9L tumors transplanted into the subcutis of the hind leg of Nembutal-anesthetized (50 mg/kg) Fischer 344 rats. Recessed-tip oxygen microelectrodes were inserted into the tumor, and linear pO 2 measurements were recorded in 50-m steps along a 3-mm path, and blood pressure was simultaneously measured via femoral arterial access. Additionally, pO 2 was measured at a single location for 90 to 120 minutes in FSA (n ‫؍‬ 11) or 9L tumors (n ‫؍‬ 12). Rats were switched from air to 100% O 2 breathing after 45 minutes. Temporal pO 2 records were evaluated for their potential radiobiological significance by assessing the number of times they crossed a 10-mm-Hg threshold. In addition, the data were subjected to Fourier analysis for air and O 2 breathing.FSA and 9L tumors had spatial median pO 2 measurements of 4 and 1 mm Hg, respectively. 9L had more low pO 2 measurements <2.5 mm Hg than did FSA, whereas between 2.5 and 10 mm Hg this pattern was reversed. Pimonidazole staining patterns in FSA and 9L tumors supported these results. Temporal pO 2 instability was observed in all experiments during air and O 2 breathing. Threshold analyses indicated that the 10 mm Hg threshold was crossed 2 to 5 times per hour, independent of tumor type. However, the magnitude of 9L pO 2 fluctuations was approximately eight times greater than FSA fluctuations, as assessed with Fourier transform analysis (Wilcoxon, P < 0.005). O 2 breathing significantly increased median pO 2 in FSA from 3 to 8 mm Hg (P < 0.005) and caused a significant increase in frequency and magnitude of pO 2 fluctuations. One hundred percent O 2 breathing had no effect on 9L tumor pO 2 , and it decreased the magnitude of pO 2 fluctuations with borderline significance.These results show that these two tumors differ significantly with respect to spatial and temporal oxygenation conditions under air and O 2 breathing. Fluctuations of pO 2 of the type reported herein are predicted to significantly affect radiotherapy response and could be a source for genetic instability, increased angiogenesis, and metastases.

Abstract-In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). Ithas been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O 2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO 2 (100% O 2 ) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues. Key Words: nitric oxide Ⅲ hemoglobin Ⅲ oxygen Ⅲ hemodynamics Ⅲ blood flow H emoglobin (Hb) of red blood cells (RBC) is a tetrameric protein composed of 2 ␣ and 2 ␤ chains, each containing a heme prosthetic group. One ␣ and 1 ␤ chain is combined in stable ␣␤ dimers, and 2 dimers are more loosely associated to form tetramers. The physiological role of Hb depends on its ability to reversibly bind O 2 at its heme iron centers. This transport capacity is governed by a cycle of allosteric transitions in which Hb assumes the R (relaxed, high O 2 affinity) conformation to bind O 2 in the lungs and, on partial deoxygenation, the T (tense, low O 2 affinity) conformation to efficiently deliver O 2 to peripheral tissues. This transition also controls the reactivity of 2 conserved cysteines on the ␤ chains (Cys␤93). Thiols of the cysteines can react with the potent vasodilator nitric oxide (NO) to form S-nitrosohemoglobin (SNO-Hb) in the R conformation, and preferentially unload SNO in the T conformation. [1][2][3] It has therefore been proposed that Hb would carry NO equivalents from the lungs to the periphery, thereby bringing tissue blood flow in line with oxygen demand. Although mechanisms are debated, 4 -7 some consensus has been reached, namely that Hb can act as an oxygen sensor and oxygen-dependent transducer of NO bioactivity, 7-9 and the direct coupling between SNO and O 2 content of Hb has been recently affirmed in intact human erythrocytes (Doctor et al, unpublished observations).Outside the red blood cell (R...