R. D. Braun

Quantification of longitudinal tissue pO2 gradients in window chamber tumours : impact on tumour hypoxia

SummaryWe previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9–11 days after transplantation, at a diameter of 3–4 mm; the tissue thickness was 200 μm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 μm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light ~50 vs >200 μm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). There was no significant difference in pO2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours.

The effects of hyperoxic and hypercarbic gases on tumour blood flow

Carbogen (95% O2 and 5% CO2) has been used in preference to 100% oxygen (O2) as a radiosensitizer, because it is believed that CO2 blocks O2-induced vasoconstriction. However, recent work suggests that both normal and tumour arterioles of dorsal flap window chambers exhibit the opposite: no vasoconstriction vs constriction for O2 vs carbogen breathing respectively. We hypothesized that CO2 content might cause vasoconstriction and investigated the effects of three O2–CO2 breathing mixtures on tumour arteriolar diameter (TAD) and blood flow (TBF). Fischer 344 rats with R3230Ac tumours transplanted into window chambers breathed either 1%, 5%, or 10% CO2 + O2. Intravital microscopy and laser Doppler flowmetry were used to measure TAD and TBF respectively. Animals breathing 1% CO2 had increased mean arterial pressure (MAP), no change in heart rate (HR), transient reduction in TAD and no change in TBF. Rats breathing 5% CO2 (carbogen) had transiently increased MAP, decreased HR, reduced TAD and a sustained 25% TBF decrease. Animals exposed to 10% CO2 experienced a transient decrease in MAP, no HR change, reduced TAD and a 30–40% transient TBF decrease. The effects on MAP, HR, TAD and TBF were not CO2 dose-dependent, suggesting that complex physiologic mechanisms are involved. Nevertheless, when ≥ 5% CO2 was breathed, there was clear vasoconstriction and TBF reduction in this model. This suggests that the effects of hypercarbic gases on TBF are site-dependent and that use of carbogen as a radiosensitizer may be counterproductive in certain situations.

Effects of diethylamine/nitric oxide on blood perfusion and oxygenation in the R3230Ac mammary carcinoma

The effects of intravenous diethylamine/nitric oxide (DEA/NO), a short-acting nitric oxide (NO) donor, on systemic haemodynamics, muscle and tumour blood flow (MBF and TBF) and tumour oxygenation were examined in rats bearing subcutaneous R3230Ac carcinoma in the leg. The effects of DEA/NO on the diameters of tumour-feeding and normal arterioles were evaluated in window chambers with and without implanted tumours. DEA/NO reduced mean arterial pressure (MAP) when given at doses > or = 100 nmol kg(-1), with maximal suppression at 0.5-1 min followed by return to baseline within 20 min. DEA/NO did not affect MBF except at the highest doses (500 and 1000 nmol kg(-1)). In contrast, DEA/NO reduced TBF and constricted tumour arterioles at doses > or = 100 nmol kg(-1). Tumour arteriolar vasomotion occurred in more than half the animals during hypotension and with a significantly higher frequency than in normal granulating tissue at a dose of 500 nmol kg(-1). Normal arterioles rapidly and significantly vasodilated for about 3 min and then returned to baseline. The reductions in TBF and MAP were accompanied by synchronous reduction in tumour pO2. Our findings suggest that DEA/NO decreases TBF in two ways. In the window chamber model, vascular steal occurs as normal arterioles adjacent to tumour dilate more than tumour arterioles during the initial period of hypotension. In leg tumours, the predominant mechanism is attributable to reduced perfusion pressure induced by lowered MAP, which decreases flow to the tumour, probably because of relatively higher flow resistance. The vasoconstriction and vasomotion in tumour arterioles during DEA/NO-induced hypotension may reflect differences in regulatory metabolism of NO between neoplastic and normal arterioles. Thus, intravenous injection of a short-acting NO donor, DEA/NO, decreases MAP and heart rate, leading to subsequent decreases in tumour blood flow and oxygenation.

Oxygen Regulation of Tumor Perfusion by S-Nitrosohemoglobin Reveals a Pressor Activity of Nitric Oxide

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has 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% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) 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.

Local 42°C Hyperthermia Improves Vascular Conductance of the R3230Ac Rat Mammary Adenocarcinoma during Sodium Nitroprusside Infusion

Abstract Meyer, R. E., Braun, R. D., Rosner, G. L. and Dewhirst, M. W. Local 42°C Hyperthermia Improves Vascular Conductance of the R3230Ac Rat Mammary Adenocarcinoma during Sodium Nitroprusside Infusion. The effect of sodium nitroprusside-induced hypotension on the perfusion of the R3230 adenocarcinoma during local 42°C hyperthermia was studied using a combination of intravital microscopy and laser Doppler flowmetry. Fischer 344 rats were implanted with dorsal skin flap window chambers containing the R3230Ac tumor and allocated to three treatment groups (34°C with nitroprusside, 42°C with nitroprusside, and 42°C with 0.9% saline). After baseline observation at 34°C, tumors were locally heated to 42°C using a water bath and either 0.9% saline or nitroprusside sufficient to reduce blood pressure 20% below pretreatment baseline was infused. Nitroprusside at 34°C decreased tumor vascular conductance 40% with no effect on the diameter of arterioles entering the tumor. The diameter of arterioles entering 42°C heated tumors increased 35% independent of blood pressure change. Saline at 42°C had no effect on tumor vascular conductance; however, nitroprusside at 42°C increased tumor vascular conductance 55%. Local 42°C tumor heating, combined with a moderate reduction in blood pressure with nitroprusside, overrides the vascular steal effect associated with reduced perfusion pressure alone and results in improved tumor perfusion. Observations of the effect of vasodilator substances on normothermic tumor perfusion cannot be extrapolated to situations where moderate hyperthermia is used.

Oxygen microelectrode measurements in R3230ac tumors during photodynamic therapy with verteporfin

Measurements of tissue oxygen partial pressure (pO2), blood flow and blood pressure were recorded in rat mammary R323OAc tumors during treatment with verteporfin-based photodynamic therapy. Microelectrodes of 12 micron tip diameter were used in a fixed position throughout the measurements to provide data on the temporal changes at a single location within the tissue. The microelectrode signal changes in P°2 indicated an overall trend to acute loss of P°2 after treatment, but with some notable exceptions where the P02 did not decrease either during the treatment or after treatment. These regions which showed little change in P02 were correlated with locations in which the P°2 was high to begin with before the initiation of treatment. Blood flow decreased during treatment, with the dominant effect immediately upon the initiation of treatment.