Analysis of the heterogeneity of pO2 dynamics during photodynamic therapy with verteporfin.

Photodynamic therapy (PDT) with verteporfin provides a reliable way to destroy malignant tissues. Changes in the blood flow and oxygen partial pressure (pO2) during verteporfin-PDT were studied here in the tumor tissue of the rat mammary R3230Ac carcinoma model. Oxygen microelectrodes (6-12 microns tip diameter) were used to measure the transients locally within tumors during intravenous injection of 1.0 mg/kg verteporfin followed by irradiation 15 min later with 690 nm light at 200 mW/cm2, for a cumulative dose of 144 J/cm2. The observed changes in pO2 were heterogeneous and there was a difference in the response of low-pO2 regions relative to higher-pO2 regions. The change in pO2 in hypoxic tissue regions (pO2 < 8 mmHg) had acute pO2 loss after treatment, whereas the response in regions of higher pO2 (> 8 mm Hg) was more heterogeneous with some areas maintaining their pO2 value after treatment was completed. Blood flow measurements taken on a subset of the animals indicated a significant loss in flow during the initial light delivery that remained low after treatment, indicating some vascular stasis. The results suggest that hypoxic or poorly perfused vessels may be more susceptible to acute stasis than normoxic vessels in this treatment protocol.

Tissue gradients of energy metabolites mirror oxygen tension gradients in a rat mammary carcinoma model.

PURPOSE: It has been shown that oxygen gradients exist in R3230AC tumors grown in window chambers. The fascial surface is better oxygenated than the tumor surface. The purpose of the present study was to determine whether gradients exist for energy metabolites and other end points related to oxygen transport. METHODS AND MATERIALS: Imaging bioluminescence was used to measure ATP, glucose, and lactate in cryosections of R3230AC tumors. Mean vessel density and hypoxic tissue fraction were assessed using immunohistochemistry. Tumor redox ratio was assessed by redox ratio scanning. RESULTS: Lactate content and hypoxic fraction increased, whereas ATP, glucose, redox ratio, and vessel density decreased from the fascial to the tumor surface. CONCLUSIONS: The data support a switch from aerobic to anaerobic metabolism concomitant with the PO2 gradient. The vascular hypoxia that exists in perfused vessels at the tumor surface leads to macroscopic tissue regions with restricted oxygen availability and altered metabolic status. Methods to reduce tumor hypoxia may have to take this into account if such gradients exist in human tumors. The results also have implications for hypoxia imaging, because macroscopic changes in PO2 (or related parameters) will be easier to see than PO2 gradients limited to the diffusion distance of oxygen.

Simultaneous administration of glucose and hyperoxic gas achieves greater improvement in tumor oxygenation than hyperoxic gas alone.

PURPOSE: To test the feasibility of hyperglycemic reduction of oxygen consumption combined with oxygen breathing (O(2)), to improve tumor oxygenation. METHODS AND MATERIALS: Fischer-344 rats bearing 1 cm R3230Ac flank tumors were anesthetized with Nembutal. Mean arterial pressure, heart rate, tumor blood flow ([TBF], laser Doppler flowmetry), pH, and pO(2) were measured before, during, and after glucose (1 or 4 g/kg) and/or O(2). RESULTS: Mean arterial pressure and heart rate were unaffected by treatment. Glucose at 1 g/kg yielded maximum blood glucose of 400 mg/dL, no change in TBF, reduced tumor pH (0.17 unit), and 3 mm Hg pO(2) rise. Glucose at 4 g/kg yielded maximum blood glucose of 900 mg/dL, pH drop of 0.6 unit, no pO(2) change, and reduced TBF (31%). Oxygen tension increased by 5 mm Hg with O(2). Glucose (1 g/Kg) + O(2) yielded the largest change in pO(2) (27 mm Hg); this is highly significant relative to baseline or either treatment alone. The effect was positively correlated with baseline pO(2), but 6 of 7 experiments with baseline pO(2) < 10 mm Hg rose above 10 mm Hg after combined treatment. CONCLUSION: We demonstrated the feasibility of combining hyperglycemia with O(2) to improve tumor oxygenation. However, some cell lines are not susceptible to the Crabtree effect, and the magnitude is dependent on baseline pO(2). Additional or alternative manipulations may be necessary to achieve more uniform improvement in pO(2).

Comparison of tumor and normal tissue oxygen tension measurements using OxyLite or microelectrodes in rodents.

In this study we compare oxygen tension (PO2) histograms measured with O2 microelectrodes and a new optical PO2 measurement device, the OxyLite, in normal tissues (mouse spleen and thymus) and in tumors (R3230Ac in rats) (n = 5-6). The transient response to glucose infusion or 100% O2 breathing (hyperoxia) was also measured in tumors. PO2 histograms of spleen and thymus with the two devices were not different. The OxyLite tumor PO2 histogram, however, was left-shifted compared with the microelectrode (median PO2 1.0 vs. 4.0 mmHg, P = 0.016). Both probes responded to acute hyperglycemia with a mean increase of 3-6 mmHg, but the microelectrode change was not significant. The OxyLite consistently recorded large PO2 increases (approximately 28 mmHg) with hyperoxia, whereas the microelectrode response was variable. The OxyLite averages PO2 over an area that contains interstitial and vascular components, whereas the microelectrode measures a more local PO2. This study demonstrates the importance of considering the features of the measurement device when studying tissues with heterogeneous PO2 distributions (e.g., tumors).

Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature.

The efficacy of novel cancer therapeutics can be hampered by inefficient delivery of agents to the tumor at effective concentrations. Liposomes have been used as a method to overcome some delivery issues and, in combination with hyperthermia, have been shown to increase drug delivery to tumors. This study investigates the effects of a range of temperatures (34-42 degrees C) and hyperthermia treatment scheduling (time between hyperthermia and drug administration as well as between consecutive hyperthermia treatments) on the extravasation of nanoparticles (100-nm liposomes) from tumor microvasculature in a human tumor (SKOV-3 ovarian carcinoma) xenograft grown in athymic nude mouse window chambers. Under normothermic conditions (34 degrees C) and at 39 degrees C, nanoparticles were unable to extravasate into the tumor interstitium. From 40 to 42 degrees C, nanoparticle extravasation increased with temperature, reaching maximal extravasation at 42 degrees C. Temperatures higher than 42 degrees C led to hemorrhage and stasis in tumor vessels. Enhanced nanoparticle extravasation was observed several hours after heating, decaying back to baseline at 6 h postheating. Reheating (42 degrees C for 1 h) 8 h after an initial heating (42 degrees C for 1 h) did not result in any increased nanoparticle extravasation, indicating development of vascular thermotolerance. The results of this study have implications for the application and scheduling of hyperthermia combined with other therapeutics (e.g., liposomes, antibodies, and viral vectors) for the treatment of cancer.

In vivo BOLD contrast MRI mapping of subcutaneous vascular function and maturation: validation by intravital microscopy.

Bold contrast MRI was applied for mapping vascular maturation in tumor- and wound-induced skin angiogenesis using the response of mature vessels to hypercapnia (inhalation of air vs. air 5% CO(2)) and the response of all vessels to hyperoxia (air 5% CO(2) vs. oxygen 5% CO(2) (carbogen)). MRI signal enhancement with hypercapnia was reduced in centered vs. linear phase encoding, suggesting increased blood flow. However, intravital microscopy demonstrated constriction of arterioles and reduced flux and density of red blood cells in mature capillaries with hypercapnia, with no change in the diameter of wound-induced neovasculature. The discrepancy in flow between MRI and intravital microscopy is consistent with increased plasma flow and reduced hematocrit. Hyperoxia resulted in increased blood oxygenation and constriction of all vessels. These results provide a hemodynamic explanation for the selective registration of MRI response to hypercapnia with mature vessels and the response to hyperoxia with total vascular function.

Effects of the interaction between carbogen and nicotinamide on R3230 Ac tumor blood flow in Fischer 344 rats.

Braun, R. D., Lanzen, J. L., Turnage, J. A., Rosner, G. and Dewhirst, M. W. Effects of the Interaction between Carbogen and Nicotinamide on R3230 Ac Tumor Blood Flow in Fischer 344 Rats. Radiat. Res. 155, 724-733 (2001). The purpose of this study was to determine whether there are interactions between carbogen breathing and various doses of nicotinamide at the level of the tumor arteriole that might contribute to the improvement in tumor blood flow and pO(2) that is often seen with this combination treatment. R3230 adenocarcinomas were implanted and grown to 4-5 mm in dorsal skin flap window chambers in F344 rats. Saline or 65, 200 or 500 mg/kg nicotinamide was injected i.p. while the rat breathed air through a face mask. After 20 min, either the breathing gas was switched to carbogen for 60 min or the animal remained on air. Measured end points included diameter of tumor arterioles, tumor perfusion, mean arterial blood pressure, and heart rate. None of the measured parameters were affected by injection of saline or nicotinamide, except at the highest nicotinamide dose (500 mg/kg). Mean arterial blood pressure showed a median decrease of 25% when 500 mg/kg nicotinamide was given. Diameter of tumor arterioles decreased significantly from 5-15 min after 500 mg/kg nicotinamide was given but was back to baseline by 20 min. Blood flow decreased significantly 5-20 min after administration of 500 mg/kg nicotinamide compared to the baseline prior to injection. Carbogen breathing resulted in a small increase in mean arterial blood pressure in all groups. There was a transient decrease in the diameter of tumor arterioles and blood flow during the first 5 min of carbogen breathing that was statistically significant in several groups. In the group injected with 500 mg/kg nicotinamide, the diameter of tumor arterioles increased by about 10% during the first 25 min of carbogen breathing, and blood flow increased by a median of 75% over the level prior to carbogen breathing up to 40 min after carbogen breathing. The increase in flow in this group was most likely caused by the concomitant arteriolar vasodilation. Thus there was direct evidence for an interaction between carbogen breathing and nicotinamide, but only at the dose of 500 mg/kg nicotinamide. Since this dose yields plasma levels of nicotinamide that are higher than can be tolerated clinically, it is uncertain whether these changes in arteriolar diameter and blood flow would occur in human tumors.

Review of methods used to study oxygen transport at the microcirculatory level.

The existence of hypoxic regions in tumors has long been recognized as a key factor leading to radiation resistance. More recently, it has been found that low oxygen levels also affect drug resistance, angiogenesis, cytokine production, cell cycle control, apoptosis, and metastatic propensity of tumors. Until now, most approaches to eliminating hypoxia have been empirical. However, improved understanding of the underlying mechanisms may permit the development of more soundly based, effective approaches. As discussed in this review, critical evaluation of the factors governing oxygen transport in tumors requires a thorough understanding of the methods used to study this process. Many experimental methodologies can be used to address these issues. In this review, the emphasis is placed on techniques that measure parameters on the scale of the diffusion distance of oxygen. Studies at the microregional level provide the most detailed physiological information on such processes. Over the past few years, significant progress in technology has allowed us to measure tumor oxygenation, yet spatial and temporal heterogeneities continue to provide significant challenges to obtaining clear knowledge of oxygen transport.

Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size.

The efficacy of novel cancer therapeutics has been hampered by the ability to deliver these agents to the tumor at effective concentrations. Liposomes have been used as a method to overcome some delivery issues and, in combination with hyperthermia, have been shown to increase drug delivery to tumors. Particle size has been shown to affect the delivery of liposomes, but it is not known how hyperthermia affects size dependence. This study investigates the effect of hyperthermia (42 degrees C) on the extravasation of different sized nanoparticles (albumin; 100-, 200-, and 400-nm liposomes) from tumor microvasculature in a human tumor (SKOV-3 ovarian carcinoma) xenograft grown in mouse window chambers. In this model (at 34 degrees C), no liposomes were able to extravasate into the tumor interstitium. Hyperthermia enabled liposome extravasation of all sizes. The magnitude of hyperthermia-induced extravasation was inversely proportional to particle size. Thus, at normothermia (34 degrees C), the pore cutoff size for this model was between 7 and 100 nm (e.g., liposomes did not extravasate). At 42 degrees C, the pore cutoff size was increased to >400 nm, allowing all nanoparticles tested to be delivered to the tumor interstitium to some degree. With hyperthermia, the 100-nm liposome experienced the largest relative increase in extravasation from tumor vasculature. Hyperthermia did not enable extravasation of 100-nm liposomes from normal vasculature, potentially allowing for tumor-specific delivery. These experiments indicate that hyperthermia can enable and augment liposomal drug delivery to tumors and potentially help target liposomes specifically to tumors.

Local 42 degrees 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 degrees 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 degrees C with nitroprusside, 42 degrees C with nitroprusside, and 42 degrees C with 0.9% saline). After baseline observation at 34 degrees C, tumors were locally heated to 42 degrees 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 degrees C decreased tumor vascular conductance 40% with no effect on the diameter of arterioles entering the tumor. The diameter of arterioles entering 42 degrees C heated tumors increased 35% independent of blood pressure change. Saline at 42 degrees C had no effect on tumor vascular conductance; however, nitroprusside at 42 degrees C increased tumor vascular conductance 55%. Local 42 degrees 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.

Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models.

BACKGROUND: There is a paucity of information about events that follow immediately after tumor cells are triggered to initiate the process of angiogenesis (the formation of new blood vessels). Such information is relevant to the issue of when micrometastases vascularize and has implications for the accessibility of micrometastases to various treatments. In this study, we attempted to monitor events at the initiation of angiogenesis at the earliest possible stage of tumor growth in vivo. METHODS: Two different rodent mammary tumor cell lines, R3230Ac from the Fischer 344 rat and 4T1 from the BALB/c mouse, were stably transfected with a gene that encodes an enhanced version of green fluorescence protein (GFP). GFP-labeled R3230Ac or 4T1 cells (about 20-50 cells) were implanted into dorsal skinfold window chambers of Fischer 344 rats or BALB/c mice, respectively. Tumor angiogenesis was then monitored serially and noninvasively for up to 4 weeks. RESULTS: Clear evidence of modification of the host vasculature was observed when tumor mass reached approximately 60-80 cells, and functional new blood vessels were seen when tumor mass reached roughly 100-300 cells. Individual tumor cells exhibited a chemotaxis-like growth pattern toward the pre-existing host vasculature. When ex-flk1 (a soluble, truncated vascular endothelial cell growth factor receptor protein known to be antiangiogenic) was injected with the tumor cells, the initial angiogenic and tumor growth activities were inhibited considerably, indicating that angiogenesis inhibitors may halt tumor growth even before the onset of angiogenesis. CONCLUSION: Angiogenesis induced by tumor cells after implantation in the host begins at a very early stage, i.e., when the tumor mass contains roughly 100-300 cells. Identification of chemotactic signals that initiate tumor cell migration toward the existing vasculature may provide valuable targets for preventing tumor progression and/or metastases.

Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release.

The tumor drug concentrations, drug distributions, and therapeutic efficacies achieved by three fundamentally different liposomes, nonthermosensitive liposome (NTSL), traditional thermosensitive liposome (TTSL), and low temperature sensitive liposome (LTSL); free doxorubicin (DOX); and saline in combination with hyperthermia (HT) were directly compared in a human tumor xenograft model. NTSL is a nonthermosensitive liposome in the physiological temperature range, TTSL is a traditional thermosensitive liposome that triggers in the range of approximately 42-45 degrees C and releases drug over approximately 30 min, and LTSL is a new low temperature sensitive liposome that triggers in the range of approximately 39-40 degrees C and releases drug in a matter of seconds. Because of the different attributes of the liposomes, it was possible to delineate the relative importance of liposome drug encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and HT-triggered liposomal drug release in achieving antitumor activity. Athymic nude mice bearing the FaDu human tumor xenograft were given a single i.v. dose of 5 mg/kg of DOX (free drug or liposome encapsulated), and the tumors were then heated to either 34 degrees C or 42 degrees C for 1 h at 34 degrees C. All treatment groups were similar, achieving low concentrations of DOX (0-4.5 ng/mg). At 42 degrees C, the LTSL (25.6 ng/mg) achieved the highest DOX concentration (P < 0.04), but all three liposomal formulations (7.3-25.6 ng/mg) were higher than saline or DOX (0-0.7 ng/mg; P < 0.02). LTSL + HT was also the only group that resulted in significant amounts of DNA-bound DOX (silver nitrate-extractable fraction; P < 0.02). Tumor tissue sections were visualized for DOX fluorescence to investigate the local distribution of the drug in the tumor and confirm the relative drug concentrations based on fluorescence intensity. There was relatively little fluorescence seen with treatment groups at 34 degrees C. At 42 degrees C, the LTSL showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive throughout the tumor sections. Therapeutic efficacy of treatments was determined from tumor growth time. At 34 degrees C, the only treatment group significantly better than the saline group (9.8 days) was the NTSL group, with a growth time of 20.9 days (P < 0.02). At 42 degrees C, all three liposomal formulations were more efficacious than DOX. LTSL + HT had the longest growth time (51.4 days) and the most number of local controls at 60 days (six of nine tumors). With HT, the DOX concentrations and fluorescence were tightly correlated with tumor growth delay, indicating that adequate (increased) drug delivery can be predictive of therapeutic effect. Overall, the LTSL + HT group showed the largest DOX concentration, the highest and most pervasive DOX fluorescence, and the most antitumor effect. Thus, HT-triggered liposomal drug release may account for the largest differential therapeutic effect and demonstrates the importance of rapid drug release from the drug carriers at the tumor site.

Fourier analysis of fluctuations of oxygen tension and blood flow in R3230Ac tumors and muscle in rats.

Tumor hypoxia is a major barrier to tumor radiation therapy. Typically tumor hypoxia occurs in two forms: chronic and acute. Although the existence of acute hypoxia has long been acknowledged, its temporal characteristics have never been directly measured and documented. In this study tumor PO(2), blood flow (BF), and arterial blood pressure (BP) were measured simultaneously in nine Fischer 344 rats bearing R3230Ac rat mammary adenocarcinomas in the subcutis of the left hindleg. We measured PO(2) at a single location for 36-125 min using recessed-tip oxygen microelectrodes. Simultaneously, we measured tumor BF at two sites within the tumor using laser-Doppler flowmetry (LDF). Similar recordings were made in the quadriceps muscle of seven non-tumor-bearing rats. The PO(2), tumor BF, and BP records were subjected to Fourier analysis. PO(2) and BF showed low-frequency fluctuations (<2 cycles/min) in both tumor and muscle, but the magnitude of the changes in tumor was greater. Tumor BF showed more activity at low frequencies than muscle BF, and the magnitude tended to be greater. No strong correlations were found between PO(2) and BF power spectra for either tumor or muscle or between the frequency patterns of BP and tumor PO(2) spectra. These results quantitatively demonstrate, for the first time, that BF and PO(2) fluctuate at very low frequencies in tumors. In addition to having biological significance for tumor therapy, these fluctuations may have the potential to alter tumor cell behavior via induction of hypoxia reoxygenation injury and/or altered gene expression.

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 > or = 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.

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

We 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 microm. 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 microm. 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 approximately 50 vs >200 microm 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.

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals.

One of the earliest pathologic changes of diabetes mellitus is increased nonenzymatic glycosylation (i.e., glycation) of proteins, which results in abnormal aggregation of collagen fibrils and production of superoxide radicals. These abnormalities may be responsible for the precocious senescence of connective tissue associated with the disease. We sought to determine whether glycation is increased in the vitreous humor of short-term diabetic cats (6 months' duration) and rabbits (2 months' duration), using a nitroblue tetrazolium colorimetric assay for fructosamine. Vitreous protein fructosamine concentration was significantly higher in diabetic cats and rabbits, compared with that in control (nondiabetic) animals. These results indicate that glycation is increased in the vitreous humor of short-term diabetic animals, and therefore may be one of the initial triggers for clinically apparent diabetic retinopathy.

Temporal changes in PO2 of R3230AC tumors in Fischer-344 rats.

PURPOSE: The purpose of this study was to characterize the kinetics of hypoxia-reoxygenation in a murine tumor. Information on the prevalence and kinetics of this process are lacking in solid tumors, although there are data on blood flow fluctuation. MATERIALS AND METHODS: Oxygen tension (pO2) was monitored at one position in 1 cm diameter R3230Ac tumors of Fischer-344 rats, using 10-12 microm diameter recessed-tip polarographic electrodes. Data were collected continuously at a sampling frequency of 25 Hz for 30-90 min. Mean arterial blood pressure (MAP) and heart rate were also monitored. RESULTS: Temporal fluctuations in pO2 were observed in all 13 experiments. To assess the potential for hypoxia-reoxygenation, two threshold pO2 values were chosen (5 and 10 mmHg), and the number and duration of intervals that measurements resided below the thresholds was quantitated. In some experiments, the measurements did not fluctuate across the threshold values and, instead, either remained above or below them throughout the observation period. The percentage of sites that did not fluctuate across the thresholds was 38 and 61% for the 10- and 5-mmHg values, respectively. For the remaining studies, fluctuations above and below the thresholds of hypoxia ranged around 4-7 events per h. There were wide variations in the duration of hypoxic episodes, ranging from less than 1 to more than 40 min. The percentage time that measurements were below the hypoxic thresholds was also variable, ranging from 30-90%. CONCLUSIONS: These results, taken with the already published data on temporal instability in human and murine tumor blood flow, suggest that intermittent hypoxia is a common phenomenon in tumors. Future studies will focus on the underlying mechanisms that contribute to this process, because it has important implications for radiation and chemotherapy and, perhaps, gene regulation in tumors.