Huagang Hou

The effects of ketamine–xylazine anesthesia on cerebral blood flow and oxygenation observed using nuclear magnetic resonance perfusion imaging and electron paramagnetic resonance oximetry

Ketamine-xylazine is a commonly used anesthetic for laboratory rats. Previous results showed that rats anesthetized with ketamine-xylazine can have a much lower cerebral partial pressure of oxygen (P(t)O(2)), compared to unanesthetized and isoflurane anesthetized rats. The underlying mechanisms for the P(t)O(2) reduction need to be elucidated. In this study, we measured regional cerebral blood flow (CBF) using nuclear magnetic resonance (NMR) perfusion imaging and cortical P(t)O(2) using electron paramagnetic resonance (EPR) oximetry in the forebrain of rats under isoflurane, ketamine, ketamine-xylazine and isoflurane-xylazine anesthesia. The results show that in ventilated rats ketamine at a dose of 50 mg/kg does not induce significant changes in CBF, compared to isoflurane. Ketamine-xylazine in combination causes 25-65% reductions in forebrain CBF in a region-dependent manner. Adding xylazine to isoflurane anesthesia results in similar regional reductions in CBF. EPR oximetry measurements show ketamine increases cortical P(t)O(2) while xylazine decreases cortical P(t)O(2). The xylazine induced reduction in CBF could explain the reduced brain oxygenation observed in ketamine-xylazine anesthetized rats.

Changes in Oxygenation of Intracranial Tumors With Carbogen: a BOLD MRI and EPR Oximetry Study

To examine, using blood oxygen level dependent (BOLD) MRI and EPR oximetry, the changes in oxygenation of intracranial tumors induced by carbogen breathing.

The role of oxygen during fracture healing

Oxygen affects the activity of multiple skeletogenic cells and is involved in many processes that are important for fracture healing. However, the role of oxygen in fracture healing has not been fully studied. Here we systematically examine the effects of oxygen tension on fracture healing and test the ability of hyperoxia to rescue healing defects in a mouse model of ischemic fracture healing. Mice with tibia fracture were housed in custom-built gas chambers and groups breathed a constant atmosphere of 13% oxygen (hypoxia), 21% oxygen (normoxia), or 50% oxygen (hyperoxia). The influx of inflammatory cells to the fracture site, stem cell differentiation, tissue vascularization, and fracture healing were analyzed. In addition, the efficacy of hyperoxia (50% oxygen) as a treatment regimen for fracture nonunion was tested. Hypoxic animals had decreased tissue vascularity, decreased bone formation, and delayed callus remodeling. Hyperoxia increased tissue vascularization, altered fracture healing in un-complicated fractures, and improved bone repair in ischemia-induced delayed fracture union. However, neither hypoxia nor hyperoxia significantly altered chondrogenesis or osteogenesis during early stages of fracture healing, and infiltration of macrophages and neutrophils was not affected by environmental oxygen after bone injury. In conclusion, our results indicate that environmental oxygen levels affect tissue vascularization and fracture healing, and that providing oxygen when fractures are accompanied by ischemia may be beneficial.

Seizure‐induced formation of isofurans: novel products of lipid peroxidation whose formation is positively modulated by oxygen tension

We have previously shown that seizures induce the formation of F2‐isoprostanes (F2‐IsoPs), one of the most reliable indices of oxidative stress in vivo. Isofurans (IsoFs) are novel products of lipid peroxidation whose formation is favored by high oxygen tensions. In contrast, high oxygen tensions suppress the formation of F2‐IsoPs. The present study determined seizure‐induced formation of IsoFs and its relationship with cellular oxygen levels (pO2). Status epilepticus (SE) resulted in F2‐IsoP and IsoF formation, with overlapping but distinct time courses in hippocampal subregions. IsoF, but not F2‐IsoP formation coincided with mitochondrial oxidative stress. SE resulted in a transient decrease in hippocampal pO2 measured by in vivo electron paramagnetic resonance oximetry suggesting an early phase of seizure‐induced hypoxia. Seizure‐induced F2‐IsoP formation coincided with the peak hypoxia phase, whereas IsoF formation coincided with the ‘reoxygenation’ phase. These results demonstrate seizure‐induced increase in IsoF formation and its correlation with changes in hippocampal pO2 and mitochondrial dysfunction.

Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo.

BackgroundChk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.MethodsGrowth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.ResultsMK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.ConclusionsThere are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.

The effect of oxygen therapy on brain damage and cerebral pO(2) in transient focal cerebral ischemia in the rat.

We examined the effect of hyperbaric oxygen (HBO) and normobaric oxygen (NBO) on neurologic damage and brain oxygenation before and after focal cerebral ischemia in rats. A middle cerebral artery occlusion (MCAO)/reperfusion rat model was used. The rats were sacrificed 22 h after reperfusion, and the infarct volume was evaluated. In study A, HBO (2.0 ATA), NBO (100% oxygen) and normobaric air (NBA) were each administered for 60 min in five different rat groups. The sizes of the infarcts after HBO and NBO applied during ischemia were 8.8 +/- 2.8% and 22.8 +/- 3.7% respectively of the ipsilateral non-occluded hemisphere. The infarct size after HBO applied during ischemia was statistically smaller than for NBO and NBA exposure (p < 0.01). In study B, cerebral pO(2) was measured before and after MCAO and HBO exposure (2.0 ATA for 60 min) in six rats using electron paramagnetic resonance (EPR) oximetry. The pO(2) in the ischemic hemisphere fell markedly following ischemia, while the pO(2) in the contralateral hemisphere remained within the normal range. Measurements of the pO(2) performed minutes after HBO exposure did not show an increase in the ischemic or normal hemispheres. The mean relative infarct size was consistent with the changes observed in study A. These data confirm the neuroprotective effects of HBO in cerebral ischemia and indicate that in vivo EPR oximetry can be an effective method to monitor the cerebral oxygenation after oxygen therapy for ischemic stroke. The ability to measure the pO(2) in several sites provides important information that should help to optimize the design of hyperoxic therapies for stroke.

Simultaneous measurement of rat brain cortex PtO2 using EPR oximetry and a fluorescence fiber-optic sensor during normoxia and hyperoxia

Electron paramagnetic resonance (EPR) oximetry is a promising, relatively non-invasive method of monitoring tissue partial pressure of oxygen (PtO(2)) that has proven useful in following changes in PtO(2) under various physiologic and pathophysiologic conditions. Optimal utilization of the method will be facilitated by systematic comparisons with other available methods. Here, we report on the absolute values and changes of rat brain PtO(2) using EPR oximetry and the OxyLite, an oxygen monitor based on fluorescence quenching, at adjacent locations in the same brain. EPR oximetry utilizes an implanted oxygen-sensitive material and reports tissue PtO(2) at the surface of the material. OxyLite measures PtO(2) using the fluorescence lifetime of a chromophore fixed to the tip of an optical fiber that is inserted into tissue. Measurements were made at a depth of 2-3 mm into the cortex during normoxia and during breathing of carbogen (95% O(2):5% CO(2)) followed by a return to normoxia. We conclude that in this study (1) PtO(2) values reported by the two methods are similar but not exactly the same, (2) both methods can record a baseline and rapid changes in PtO(2), (3) changes in PtO(2) induced by increasing FiO(2) from 0.26 to 0.95 (carbogen) were similar by the two methods and (4) in some rats breathing carbogen, absolute values of PtO(2) were above the sensitive range of the OxyLite method.

Clinical EPR: Unique Opportunities and Some Challenges

Electron paramagnetic resonance (EPR) spectroscopy has been well established as a viable technique for measurement of free radicals and oxygen in biological systems, from in vitro cellular systems to in vivo small animal models of disease. However, the use of EPR in human subjects in the clinical setting, although attractive for a variety of important applications such as oxygen measurement, is challenged with several factors including the need for instrumentation customized for human subjects, probe, and regulatory constraints. This article describes the rationale and development of the first clinical EPR systems for two important clinical applications, namely, measurement of tissue oxygen (oximetry) and radiation dose (dosimetry) in humans. The clinical spectrometers operate at 1.2 GHz frequency and use surface-loop resonators capable of providing topical measurements up to 1 cm depth in tissues. Tissue pO2 measurements can be carried out noninvasively and repeatedly after placement of an oxygen-sensitive paramagnetic material (currently India ink) at the site of interest. Our EPR dosimetry system is capable of measuring radiation-induced free radicals in the tooth of irradiated human subjects to determine the exposure dose. These developments offer potential opportunities for clinical dosimetry and oximetry, which include guiding therapy for individual patients with tumors or vascular disease by monitoring of tissue oxygenation. Further work is in progress to translate this unique technology to routine clinical practice.

Repeated tumor pO2 measurements by multi-site EPR oximetry as a prognostic marker for enhanced therapeutic efficacy of fractionated radiotherapy

PURPOSE To investigate the temporal effects of single or fractionated radiotherapy on subcutaneous RIF-1 tumor pO(2) and to determine the therapeutic outcomes when the timing of fractionations is guided by tumor pO(2). METHODS The time-course of the tumor pO(2) changes was followed by multi-site electron paramagnetic resonance (EPR) oximetry. The tumors were treated with single 10, 20, and 10 Gy x 2 doses, and the tumor pO(2) was measured repeatedly for six consecutive days. In the 10 Gy x 2 group, the second dose of 10 Gy was delivered at a time when the tumors were either relatively oxygenated or hypoxic. The changes in tumor volumes were followed for nine days to determine the therapeutic outcomes. RESULTS A significant increase in tumor pO(2) was observed at 24h post 10 Gy, while 20 Gy resulted in a significant increase in tumor pO(2) at 72-120 h post irradiation. The tumors irradiated with a second dose of 10 Gy at 24h, when the tumors were oxygenated, had a significant increase in tumor doubling times (DTs), as compared to tumors treated at 48 h when they were hypoxic (p<0.01). CONCLUSION Results indicate that the time of tumor oxygenation depends on the irradiation doses, and radiotherapeutic efficacy could be optimized if irradiations are scheduled at times of increased tumor oxygenation. In vivo multi-site EPR oximetry could be potentially used to monitor tumor pO(2) repeatedly during fractionated schemes to optimize radiotherapeutic outcome. This technique could also be used to identify responsive and non-responsive tumors, which will facilitate the design of other therapeutic approaches for non-responsive tumors at early time points during the course of therapy.

Tissue pO2 of Orthotopic 9L and C6 Gliomas and Tumor-Specific Response to Radiotherapy and Hyperoxygenation

PURPOSE Tumor hypoxia is a well-known therapeutic problem; however, a lack of methods for repeated measurements of glioma partial pressure of oxygen (pO(2)) limits the ability to optimize the therapeutic approaches. We report the effects of 9.3 Gy of radiation and carbogen inhalation on orthotopic 9L and C6 gliomas and on the contralateral brain pO(2) in rats using a new and potentially widely useful method, multisite in vivo electron paramagnetic resonance oximetry. METHODS AND MATERIALS Intracerebral 9L and C6 tumors were established in the left hemisphere of syngeneic rats, and electron paramagnetic resonance oximetry was successfully used for repeated tissue pO(2) measurements after 9.3 Gy of radiation and during carbogen breathing for 5 consecutive days. RESULTS Intracerebral 9L gliomas had a pO(2) of 30-32 mm Hg and C6 gliomas were relatively hypoxic, with a pO(2) of 12-14 mm Hg (p < 0.05). The tissue pO(2) of the contralateral brain was 40-45 mm Hg in rats with either 9L or C6 gliomas. Irradiation resulted in a significant increase in pO(2) of the 9L gliomas only. A significant increase in the pO(2) of the 9L and C6 gliomas was observed in rats breathing carbogen, but this effect decreased during 5 days of repeated experiments in the 9L gliomas. CONCLUSION These results highlight the tumor-specific effect of radiation (9.3.Gy) on tissue pO(2) and the different responses to carbogen inhalation. The ability of electron paramagnetic resonance oximetry to provide direct repeated measurements of tissue pO(2) could have a vital role in understanding the dynamics of hypoxia during therapy that could then be optimized by scheduling doses at times of improved tumor oxygenation.