Assessment of the changes in 9L and C6 glioma pO2 by EPR oximetry as a prognostic indicator of differential response to radiotherapy.

Tumor hypoxia impedes the outcome of radiotherapy. As the extent of hypoxia in solid tumors varies during the course of radiotherapy, methods that can provide repeated assessment of tumor pO2 such as EPR oximetry may enhance the efficacy of radiotherapy by scheduling irradiations when the tumors are oxygenated. The repeated measurements of tumor pO2 may also identify responders, and thereby facilitate the design of better treatment plans for nonresponding tumors. We have investigated the temporal changes in the ectopic 9L and C6 glioma pO2 irradiated with single radiation doses less than 10 Gy by EPR oximetry. The 9L and C6 tumors were hypoxic with pO2 of approximately 5-9 mmHg. The pO2 of C6 tumors increased significantly with irradiation of 4.8-9.3 Gy. However, no change in the 9L tumor pO2 was observed. The irradiation of the oxygenated C6 tumors with a second dose of 4.8 Gy resulted in a significant delay in growth compared to hypoxic and 2 Gy × 5 treatment groups. The C6 tumors with an increase in pO2 of greater than 50% from the baseline of irradiation with 4.8 Gy (responders) had a significant tumor growth delay compared to nonresponders. These results indicate that the ectopic 9L and C6 tumors responded differently to radiotherapy. We propose that the repeated measurement of the oxygen levels in the tumors during radiotherapy can be used to identify responders and to design tumor oxygen guided treatment plans to improve the outcome.

Chronic paracetamol treatment influences indices of reactive oxygen species accumulation in the aging Fischer 344 X Brown Norway rat aorta.

Previous reports have demonstrated that increased levels of reactive oxygen species (ROS) and alterations in cell signaling characterize aging in the Fischer 344 X Brown Norway (FBN) rat aorta. Other work has suggested that increases in ROS may be related to vascular wall thickening and the development of hypertension. Paracetamol (acetaminophen) is a potent antioxidant that has been found to diminish free radicals in ischemia-reperfusion studies. However, it remains unclear whether chronic paracetamol administration influences signaling or ROS accumulation in the aging aorta. FBN rats (27 months old; n=8) were subjected to 6 months of treatment with a therapeutic dose of paracetamol (30 mg/kg/day) and compared to age-matched untreated FBN rat controls (n=8). Compared to measurements in the aortae of 6-month old animals, tunica media thickness, tissue superoxide levels, and protein oxidation levels were 38 ± 7%, 92 ± 31%, and 7 ± 2% higher in the aortae of 33-month control animals (p ≤0.05). Chronic paracetamol treatment decreased tunica media thickness and the amount of oxidized protein by 13 ± 4% and 30 ± 1%, respectively (p ≤0.05). This finding of diminished aortic thickening was associated with increased phosphorylation (activation) of the mitogen activated protein kinases and diminished levels of the anti-apoptotic protein Bcl-2. Taken together, these data suggest that chronic paracetamol treatment may decrease the deleterious effects of aging in the FBN rat aorta.

Repeated assessment of orthotopic glioma pO(2) by multi-site EPR oximetry: a technique with the potential to guide therapeutic optimization by repeated measurements of oxygen.

Tumor hypoxia plays a vital role in therapeutic resistance. Consequently, measurements of tumor pO(2) could be used to optimize the outcome of oxygen-dependent therapies, such as, chemoradiation. However, the potential optimizations are restricted by the lack of methods to repeatedly and quantitatively assess tumor pO(2) during therapies, particularly in gliomas. We describe the procedures for repeated measurements of orthotopic glioma pO(2) by multi-site electron paramagnetic resonance (EPR) oximetry. This oximetry approach provides simultaneous measurements of pO(2) at more than one site in the glioma and contralateral cerebral tissue. The pO(2) of intracerebral 9L, C6, F98 and U251 tumors, as well as contralateral brain, were measured repeatedly for five consecutive days. The 9L glioma was well oxygenated with pO(2) of 27-36 mm Hg, while C6, F98 and U251 glioma were hypoxic with pO(2) of 7-12mm Hg. The potential of multi-site EPR oximetry to assess temporal changes in tissue pO(2) was investigated in rats breathing 100% O(2). A significant increase in F98 tumor and contralateral brain pO(2) was observed on day 1 and day 2, however, glioma oxygenation declined on subsequent days. In conclusion, EPR oximetry provides the capability to repeatedly assess temporal changes in orthotopic glioma pO(2). This information could be used to test and optimize the methods being developed to modulate tumor hypoxia. Furthermore, EPR oximetry could be potentially used to enhance the outcome of chemoradiation by scheduling treatments at times of increase in glioma pO(2).

Synergistic combination of hyperoxygenation and radiotherapy by repeated assessments of tumor pO2 with EPR oximetry.

The effect of hyperoxygenation with carbogen (95% O(2) + 5% CO(2)) inhalation on RIF-1 tumor pO(2 )and its consequence on growth inhibition with fractionated radiotherapy is reported. The temporal changes in the tumor pO(2) were assessed by in vivo Electron Paramagnetic Resonance (EPR) oximetry in mice breathing 30% O(2) or carbogen and the tumors were irradiated with 4 Gy/day for 5 consecutive days; a protocol that emulates the clinical application of carbogen. The RIF-1 tumors were hypoxic with a tissue pO(2) of 5-9 mmHg. Carbogen (CB) breathing significantly increased tumor pO(2), with a maximum increase at 22.9-31.2 min on days 1-5, however, the magnitude of increase in pO(2) declined on day 5. Radiotherapy during carbogen inhalation (CB/RT) resulted in a significant tumor growth inhibition from day 3 to day 6 as compared to 30%O(2)/RT and carbogen (CB/Sham RT) groups. The results provide unambiguous quantitative information on the effect of carbogen inhalation on tumor pO(2) over the course of 5 days. Tumor growth inhibition in the CB/RT group confirms that the tumor oxygenation with carbogen was radiobiologically significant. Repeated tumor pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcomes by scheduling doses at times of improved tumor oxygenation.

Repeated tumor oximetry to identify therapeutic window during metronomic cyclophosphamide treatment of 9L gliomas.

Malignant gliomas are aggressive and angiogenic tumors with high VEGF content. Consequently, approaches such as metronomic chemotherapy, which have an anti-angiogenic effect, are being investigated. However, a lack of an appropriate technique that can facilitate the identification of vascular changes during antiangiogenic treatments has restricted therapeutic optimization. We have investigated the potential of tumor pO2 as a marker to detect vascular changes during metronomic chemotherapy. Electron paramagnetic resonance (EPR) oximetry was used to repeatedly assess tumor pO2 during metronomic cyclophosphamide treatment of subcutaneous 9L tumors. The 9L tumors were hypoxic with a pO2 of 5.6-8 mmHg and a tumor volume of 247-300 mm3 prior to any treatment. Tumor pO2 increased significantly to 19.7 mmHg on day 10 and remained at an elevated level until day 33 during 4 weekly treatments with 140 mg/kg cyclophosphamide. A significant decrease in the tumor volume on days 21-31 occurred in the cyclophosphamide group, while the tumor volume of the control group significantly increased during measurements for two weeks. A significant tumor growth delay was achieved with two weekly treatments of cyclophosphamide plus radiotherapy (4 Gy x 5) as compared to control, cyclophosphamide and radiotherapy alone groups. The results indicate the potential of EPR oximetry to assess tumor pO2 during metronomic chemotherapy. The ability to identify the duration of an increase in tumor pO2, therapeutic window, non-invasively by EPR oximetry could have a significant impact on the optimization of antiangiogenic approaches for the treatment of gliomas. This vital information could also be used to schedule radiotherapy to enhance therapeutic outcome.

Cerebral oxygenation of the cortex and striatum following normobaric hyperoxia and mild hypoxia in rats by EPR oximetry using multi-probe implantable resonators.

Multi-site electron paramagnetic resonance (EPR) oximetry, using multi-probe implantable resonators, was used to measure the partial pressure of oxygen (pO(2)) in the brains of rats following normobaric hyperoxia and mild hypoxia. The cerebral tissue pO(2) was measured simultaneously in the cerebral cortex and striatum in the same rats before, during, and after normobaric hyperoxia and mild hypoxia challenges. The mean baseline tissue pO(2) values were not significantly different between the cortex and striatum.During 30 min of 100% O(2) inhalation, a statistically significant increase in tissue pO(2) of all four sites was observed, however, the tissue pO(2) of the striatum area was significantly higher than in the forelimb area of the cortex. Brain pO(2) significantly decreased from the baseline value during 15 min of 15% O(2) challenge.No differences in the recovery of the cerebral cortex and striatum pO(2) were observed when the rats were allowed to breathe 30% O(2). It appears that EPR oximetry using implantable resonators can provide information on pO(2) under the experimental conditions needed for such a study. The levels of pO(2) that occurred in these experiments are readily resolvable by multi-site EPR oximetry with multi-probe resonators. In addition, the ability to simultaneously measure the pO(2) in several areas of the brain provides important information that could potentially help differentiate the pO(2) changes that can occur due to global or local mechanisms.

Tumor pO2 as a surrogate marker to identify therapeutic window during metronomic chemotherapy of 9L gliomas.

Glioblastomas are aggressive and highly vascularized primary brain tumors with a 5-year survival rate of less than 10%. Approaches targeting tumor vasculature are currently being investigated to achieve therapeutic benefits for this fatal malignancy. However, lack of suitable markers that can be used to monitor therapeutic effects during such treatments has restricted their optimization. We have focused on the development of tumor pO(2) as a surrogate marker to identify the therapeutic window during metronomic chemotherapy.We report the effect of four weekly administrations of cyclophosphamide (140 mg/Kg, i.p), a chemo drug, on tumor pO(2) and growth of subcutaneous 9L tumors in SCID mice. The repeated measurement of tumor pO(2) was carried out using in vivo EPR oximetry. The subcutaneous 9L tumors were hypoxic with a pre-treatment tumor pO(2) of 5.1 ± 1 mmHg and a tumor volume of 236 ± 45 mm3 on day 0. The tumor pO(2) increased significantly to 26.2 ± 2 mmHg on day 10, and remained at an elevated level till day 31 during weekly treatments with cyclophosphamide. The tumor pO(2) then declined to 20 ± 9 mmHg on day 43. The tumor volume of the control group increased significantly with no change in tumor pO(2)over days.Results indicate a transient increase in tumor pO(2) during metronomic chemotherapy of 9L gliomas and could be potentially used as a marker to identify vessel normalization during metronomic chemotherapy. The ability to identify therapeutic window non-invasively using EPR oximetry can have a significant impact on the optimization of clinical protocols. In vivo EPR oximetry is currently being tested for repeated pO(2) measurements in patients with superficial tumors.

Effect of hyperoxygenation on tissue pO2 and its effect on radiotherapeutic efficacy of orthotopic F98 gliomas.

PURPOSE: Lack of methods for repeated assessment of tumor pO(2) limits the ability to test and optimize hypoxia-modifying procedures being developed for clinical applications. We report repeated measurements of orthotopic F98 tumor pO(2) and relate this to the effect of carbogen inhalation on tumor growth when combined with hypofractionated radiotherapy. METHODS AND MATERIALS: Electron paramagnetic resonance (EPR) oximetry was used for repeated measurements of tumor and contralateral brain pO(2) in rats during 30% O(2) and carbogen inhalation for 5 consecutive days. The T(1)-enhanced volumes and diffusion coefficients of the tumors were assessed by magnetic resonance imaging (MRI). The tumors were irradiated with 9.3 Gy x 4 fractions in rats breathing 30% O(2) or carbogen to determine the effect on tumor growth. RESULTS: The pretreatment F98 tumor pO(2) varied between 8 and 16 mmHg, while the contralateral brain had 41 to 45 mmHg pO(2) during repeated measurements. Carbogen breathing led to a significant increase in tumor and contralateral brain pO(2); however, this effect declined over days. Irradiation of the tumors in rats breathing carbogen resulted in a significant decrease in tumor growth and an increase in the diffusion coefficient measured by MRI. CONCLUSIONS: The results provide quantitative measurements of the effect of carbogen inhalation on intracerebral tumor pO(2) and its effect on therapeutic outcome. Such direct repeated pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcome by scheduling doses at times of improved tumor oxygenation. EPR oximetry is currently being tested for clinical applications.

Shear stress activates Akt during vascular smooth muscle cell reorientation.

Vascular intervention procedures can lead to endothelial damage and expose the underlying VSMCs (vascular smooth muscle cells) to shear stress. Although shear stress has been implicated in the proliferation and migration of VSMCs, the molecular mechanism(s) underlying these events are not well understood. In the present study, we examined the effect of shear stress on VSMC reorientation and the activation of Akt (also called protein kinase B) pathway signalling. Cells were subjected to a shear of 9.8 dynes/cm2 (1 dyne=10-5 N) for 0 min, 5 min, 15 min, 30 min, 1 h, 4 h and 24 h. Shear stress caused the VSMCs to realign at an angle that was approximately 45 degrees relative to the shear force vector after 24 h. Immunoblotting demonstrated that the phosphorylations of Akt and Akt-related signalling proteins [mTOR (mammalian target of rapamycin), PTEN (phosphatase and tensin homologue deleted on chromosome 10) and p70S6k (p70 S6-kinase)] were increased after shear stimulation. These results indicate that the activation of the Akt pathway signalling is closely correlated with shear-induced VSMC reorientation.

Radiotherapy in conjunction with 7-hydroxystaurosporine: a multimodal approach with tumor pO2 as a potential marker of therapeutic response.

Checkpoint inhibitors potentially could be used to enhance cell killing by DNA-targeted therapeutic modalities such as radiotherapy. UCN-01 (7-hydroxystaurosporine) inhibits S and G2 checkpoint arrest in the cells of various malignant cell lines and has been investigated in combination with chemotherapy. However, little is known about its potential use in combination with radiotherapy. We report the effect of 20 Gy radiation given in conjunction with UCN-01 on the pO2 and growth of subcutaneous RIF-1 tumors. Multisite EPR oximetry was used for repeated, non-invasive tumor pO2 measurements. The effect of UCN-01 and/or 20 Gy on tumor pO2 and tumor volume was investigated to determine therapeutic outcomes. Untreated RIF-1 tumors were hypoxic with a tissue pO2 of 5-7 mmHg. Treatment with 20 Gy or UCN-01 significantly reduced tumor growth, and a modest increase in tumor pO2 was observed in tumors treated with 20 Gy. However, irradiation with 20 Gy 12 h after UCN-01 treatment resulted in a significant inhibition of tumor growth and a significant increase in tumor pO2 to 16-28 mmHg from day 1 onward compared to the control, UCN-01 or 20-Gy groups. Treatment with UCN-01 12 h after 20 Gy also led to a similar growth inhibition of the tumors and a similar increase in tumor pO2. The changes in tumor pO2 observed after the treatment correlated inversely with the tumor volume in the groups receiving UCN-01 with 20 Gy. This multimodal approach could be used to enhance the outcome of radiotherapy. Furthermore, tumor pO2 could be a potential marker of therapeutic response.

Burn trauma in skeletal muscle results in oxidative stress as assessed by in vivo electron paramagnetic resonance.

Using a mouse model, we tested the hypotheses that severe burn trauma causes metabolic disturbances in skeletal muscle, and that these can be measured and repeatedly followed by in vivo electron paramagnetic resonance (EPR). We used a 1.2-GHz (L-band) EPR spectrometer to measure partial pressure of oxygen (pO(2)) levels, redox status and oxidative stress following a non-lethal burn trauma model to the left hind limbs of mice. Results obtained in the burned mouse gastrocnemius muscle indicated a significant decrease in tissue pO(2) immediately (P=0.032) and at 6 h post burn (P=0.004), compared to the gastrocnemius of the unburned hind limb. The redox status of the skeletal muscle also peaked at 6 h post burn (P=0.027) in burned mice. In addition, there was an increase in the EPR signal of the nitroxide produced by oxidation of the hydroxylamine (CP-H) probe at 12 h post burn injury, indicating a burn-induced increase in mitochondrial reactive oxygen species (ROS). The nitroxide signal continued to increase between 12 and 24 h, suggesting a further increase in ROS generation post burn. These results confirm genomic results, which indicate a downregulation of antioxidant genes and therefore strongly suggest the dysfunction of the mitochondrial oxidative system. We believe that the direct measurement of tissue parameters such as pO(2), redox and ROS by EPR may be used to complement measurements by nuclear magnetic resonance (NMR) in order to assess tissue damage and the therapeutic effectiveness of antioxidant agents in severe burn trauma.