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Biomedical subjects

B Klitzman

Publications and source records attributed to B Klitzman.

At least 37 records · Page 2Linked to original sources

Engineering the tissue which encapsulates subcutaneous implants. I. Diffusion properties.

This report uses normal rat subcutis as a reference point to provide a quantitative analysis of small analyte transport through the tissue which encapsulates implants. Polyvinyl alcohol (PVA) with 60- and 350-micron mean pore size (PVA-60, PVA-350), nonporous PVA (PVA-skin), and stainless-steel cage (SS) specimens were implanted in the subcutis of Sprague-Dawley rats for 4 weeks to elicit a range of capsular wound-healing tissues. Histologic examination showed that the capsular tissue which formed around PVA-skin and SS specimens was densely fibrous and avascular. That forming around PVA-60 and PVA-350 was less densely fibrous and more vascular. The fibrous content of capsular tissue and subcutis was determined from eosin-stained histologic sections. Dual-chamber diffusion measurements of sodium fluorescein (Mw 376 g/mol) through capsular tissue and normal rat subcutis were used to quantitatively compare the effective diffusion coefficients of small analytes on the order of glucose. The two most fibrous capsular tissues exhibited diffusion coefficients that were statistically (p < 0.05) less than that determined for rat subcutis by 50 and 25% for PVA-skin and SS, respectively. The diffusion coefficients of the less dense capsular tissue which formed around the porous implants were not statistically different from subcutis. The experimentally measured diffusion coefficients of the two most fibrous capsular tissues were closely predicted by a simple two-component diffusion model consisting of an aqueous interstitium with an array of impenetrable bodies equal in volume fraction to the fibrous content of the tissue. This model overestimates the diffusion coefficients measured for the least fibrous tissues. Using the diffusion coefficient measured for the PVA-skin capsular tissue, a finite difference model predicts that a 200-microns-thick capsular layer would increase from 5 to 20 min the time required for subcutaneously implanted sensor to detect 95% of the blood analyte concentration. This study suggests that the fibrous capsule forming around a subcutaneously implanted smooth-surface sensor imposes a significant diffusion barrier to small analytes such as glucose, thus increasing the lag time of the sensor by as much as threefold. A corollary observation is that a sensor with a porous surface which allows tissue ingrowth may be more responsive to blood analyte fluctuations as a result of its a more vascular and less fibrous encapsulation tissue.

Animals↗

Heparin coating of vascular prostheses reduces thromboemboli.

BACKGROUND: Synthetic conduits made from currently available materials are suboptimal for use in small-diameter vascular reconstruction because of their high surface thrombogenicity, which leads to failure. METHODS: In this study control, heparin-irrigated, or heparin-bonded expanded polytetrafluoroethylene (ePTFE) grafts (4 mm long by 1 mm inner diameter) were implanted to reconstruct the iliac artery in male rats. The cremaster muscle was isolated as an island flap based on branches of the iliac artery downstream from the graft. Emboli were quantitated by using intravital fluorescent microscopy of the cremaster muscle's microcirculation. RESULTS: The mean number of emboli observed per animal during a 20-minute period was 91 for the control group, 84 for the heparin-irrigated group, and 22 for the tridodecylmethylammonium chloride (TDMAC)-heparin group. The mean area of each embolus was 1057 microns 2 for control, 940 microns 2 for heparin-irrigated, and 808 microns 2 for TDMAC-heparin-coated grafts (p < 0.05 for TDMAC-heparin versus control or heparin-irrigated). CONCLUSIONS: A TDMAC-heparin coating of ePTFE microvascular prostheses significantly reduces downstream microemboli.

Animals↗

Laser Doppler flowmetry: a clinical test of pulpal vitality.

PROBLEM: A rapid, accurate, noninvasive method of determining pulpal blood flow would be helpful in determining pulpal vitality. OBJECTIVE: The purpose of this study was to determine if laser Doppler flowmetry can measure induced changes in pulpal blood flow. STUDY DESIGN: Two percent lidocaine with epinephrine 1:100,000 was infiltrated into the labial vestibule to anesthetize five anterior teeth in healthy human volunteers. Stents were placed on the teeth to stabilize the laser Doppler probes. Measurements of pulpal blood flow were made along with electrocardiograms to record the cardiac cycle. RESULTS: Laser Doppler flowmetry demonstrated pulpal blood flow and pulse amplitude decreases under test conditions. These decreases were most significant at 10 minutes after the injection of anesthetic with vasoconstrictor. CONCLUSIONS: Laser Doppler flowmetry was able to measure pulpal blood flow and record changes in blood flow that occurred when epinephrine was used with infiltration anesthesia. The pulse width and the mean flow were dramatically affected as was synchronization with the cardiac cycle.

Analysis of Variance↗

Iloprost in alginate decreases the thrombogenicity of expanded polytetrafluoroethylene.

Vascular prostheses of small diameter perform poorly in vivo, in part because of the high thrombogenicity of available biomaterials. This study examined the thrombogenicity of expanded polytetrafluoroethylene (ePTFE) vascular graft segments (10 mm long x 4 mm i.d.) in vitro before and after impregnation with saline, alginate, or alginate containing the stable prostacyclin analog, iloprost. Each segment was immersed in activated whole blood and the weight of the adherent thrombus was measured at specified intervals. At 6 and 7 min the saline-denucleated group accumulated significantly less thrombus than control (p < .05). Alginate alone was not significantly different from controls. The graft segments treated with alginate + iloprost accumulated significantly less thrombus (p < .05) than all other groups after 6 min. These data demonstrate that denucleation of ePTFE with iloprost in alginate dramatically decreases its in vitro thrombogenicity.

Alginates↗

Modulation of ultraviolet light-induced epidermal damage: beneficial effects of tocopherol.

Oxygen free radicals have been shown to result from and mediate deleterious effects of ultraviolet radiation on the skin. The purpose of this study was to determine if topical DL-alpha-tocopherol (vitamin E) could reduce ultraviolet-induced damage to the epidermis. Twenty mice were treated with either ethanol or a 1:1 mixture of tocopherol and ethanol. Treatments consisted of once-daily 0.1-ml topical applications for 1 week, followed by irradiation with 0.30 mW/cm2 of ultraviolet B irradiation. A statistically significant decrease in Schiff base formation was noted between tocopherol-treated animals and their controls. Histologic study revealed a statistically significant increase in epidermal thickness in tocopherol-treated skin versus controls or vehicle alone. The thicker epidermis was accompanied by the presence of parakeratosis, implicating increased proliferation as the cause of the increasing thickness. The number of sunburn cells was decreased by tocopherol treatment. Tocopherol protection from ultraviolet irradiation may have been due to both direct protection from free radicals and indirect protection by means of increased epidermal thickness. The demonstration of beneficial effects of tocopherol administration suggests that further studies in clinically relevant models to define optimal dosage, frequency of administration, vehicle, and quantitation of the possible protective effects afforded to Langerhans cells may be useful.

Administration, Topical↗

Brown recluse spider envenomation: a prospective trial of hyperbaric oxygen therapy.

OBJECTIVES: Loxosceles reclusa (brown recluse) spider bites can produce severe skin lesions that may necessitate extensive surgical repair. This study delineated the effects of hyperbaric oxygen (HBO) therapy on these lesions by performing a prospective controlled animal study. METHODS: After approval by the Institutional Animal Care and Use Committee, 41 New Zealand white rabbits received 64 intradermal injections of 73 microL of raw venom extract mixed with physiologic buffered saline (Dulbecco's solution). Control injections were made with buffer. The animals were divided into 5 groups: 1) venom and no HBO; 2) venom and 1 immediate HBO treatment (100% O2); 3) venom and immediate HBO with 10 treatments (100% O2); 4) venom and then delayed (48 hr) HBO therapy with 10 treatments (100% O2); and 5) venom and immediate hyperbaric treatment with normal inspired PO2 for 10 treatments (8.4% O2). Three animals in group 2 also received a control sodium citrate buffer injection. HBO treatments were at 2.5 atm absolute (ATA) for 90 minutes twice daily. Daily measurements were made of the lesion diameter, and skin blood flow using a laser Doppler probe. RESULTS: There was no significant effect of HBO on blood flow at the wound center or 1-2 cm from the wound center. Standard HBO significantly decreased wound diameter at 10 days (p < 0.0001; ANOVA), whereas hyperbaric treatment with normoxic gas had no effect. Histologic preparations from 2 animals in each group revealed that there were more polymorphonuclear leukocytes in the dermis of all the HBO-treated animals when compared with the venom-alone and sodium-citrate controls. CONCLUSION: HBO treatment within 48 hours of a simulated bite from L. reclusa reduces skin necrosis and results in a significantly smaller wound in this model. The mechanism appears unrelated to augmented local blood flow between treatments.

Animals↗

Pulpal response to cavity preparation with the Er:YAG and Mark III free electron lasers.

OBJECTIVE: The purpose of this investigation was to evaluate the pulp response to class V cavity preparation with the use of the Er:YAG laser and free electron laser. STUDY DESIGN: Class V cavities were prepared in 133 teeth of four beagle dogs by one of three methods: (1) Er:YAG laser, (2) free electron laser, (3) high-speed handpiece. Treatment occurred at 1 hour, 1 day, 7 days, and 28 days. The teeth were removed and the pulp evaluated. Histologically, the data were evaluated with Mantel-Haenszel analysis. RESULTS: The Er:YAG laser, free electron laser, and handpiece treatment groups resulted in specimens with normal or mild pulp reactions in 36, 46, and 42 teeth, respectively; moderate or severe reactions were observed in 7, 1, and 1 teeth, respectively. No statistically significant difference in the pulp response to the three treatment modalities was observed. CONCLUSION: The pulp response to Er:YAG laser and free electron laser application would appear to be similar to the response from high-speed handpiece application.

Animals↗

Limiting impairment of muscle function following ischemia and reperfusion in rabbits.

Reperfusion injury is a phenomenon complicating microvascular reconstruction. Extensive investigations of free-radical scavengers exist in the literature. The potential beneficial effects of verapamil, deferoxamine, and dimethylsulfoxide were studied in the rabbit forelimb subjected to 3 hr of tourniquet ischemia, followed by a period of reperfusion. Five minutes prior to tourniquet release, rabbits were given single intravenous infusions of normal saline, verapamil (0.2 mg/kg), deferoxamine (50 mg/kg), or dimethylsulfoxide (100 mg/kg). Following reperfusion, neither light microscopy nor laser Doppler flowmetry revealed significant differences between the sham and treated limbs. Muscle function was evaluated by measuring maximal twitch tensions in the flexor digitorum superficialis. Deterioration of muscle contractile function at 0.5, 3, and 24 hr after reperfusion was significantly improved by treatment with verapamil or deferoxamine. Conversely, dimethylsulfoxide worsened muscle function post ischemia. Dimethylsulfoxide does not appear to be beneficial, while verapamil and deferoxamine may be of benefit in mitigating reperfusion injury in microvascular transplantation.

Animals↗

Microsurgical composite tissue transplantation at difficult recipient sites facilitated by preliminary installation of vein grafts as arteriovenous loops.

In microvascular surgery when local recipient vessels are inadequate, vein grafting is required. There are several potential inherent disadvantages of immediate vein grafting, including the development of graft thrombosis or leakage, an increased opportunity for technical errors, and an increased number of anastomoses in series. All of these may contribute to a higher failure rate for composite-tissue transplantation requiring vein grafts. The authors hypothesized that in cases where vein grafting is obviously required, the creation of a temporary, looped, arteriovenous fistula (AVF) would reduce the morbidity of vein grafting, by allowing the detection of thrombosis or technical errors predisposing to thrombosis prior to free-tissue transplantation. Since delaying the division of an AVF for 5 or more days may allow time for healing of the endothelium at the AVF anastomotic site, the hypothesis was that composite-tissue transplantation whose vein grafts were installed as an AVF divided in a delayed manner, might have better patency than those in which vein grafts were installed at the time of reconstruction. This study reviews the results of 16 patients (8 females, 8 males) who underwent 17 microvascular reconstructions using AVFs. Patient courses and outcomes were compared between those undergoing immediate (8 patients) and delayed (7 patients) AVF construction, division, and free-tissue transplantation. There was a low patency rate for AVFs which were divided in a delayed fashion (2 of 7 patients, 29 percent), compared with those which were immediately divided (10 of 10, 100 percent). These results suggest that, in spite of a strong theoretical basis for delayed division of the AVF, delayed harvesting of the AVF is empirically associated with a higher thrombosis rate (p = 0.0048, Fishers exact test).

Adolescent↗

Assessments of thrombogenicity by three in vitro techniques. Student Research Award in the Undergraduate, Master Candidate, or Health Science Degree Candidate Category, 21st annual meeting of the Society for Biomaterials, San Francisco, CA, March 18-22, 1995.

This study assessed three in vitro techniques designed to measure the thrombogenicity of vascular grafts. All techniques immersed vascular grafts in rotating blood. In the gravimetric analysis, the weight of adherent thrombi was recorded at 2 min intervals for 20 min. In the torque analysis, a microviscometer continuously recorded the amount of torque developed as the graft rotated for 20 min. In the thrombin analysis, the blood sample was analyzed for fibrinopeptide A production indicating fibrinogen cleavage. Expanded polytetrafluoroethylene grafts were treated by removal of air nuclei (denucleation), binding of heparin, or binding of polyethylene oxide (PEO). The gravimetric analysis determined that the time at which each group experienced clot initiation was as follows: control after 6 min, denucleation after 14 min, heparin after 18 min, and PEO after 10 min. Similarly, in the torque analysis all treatment groups significantly delayed the initial increase in torque from 8.0 min for control to 12.5 min for denucleation (P < .01), > 20 min for heparin (P < .01), and 12 min for PEO (p < .05). The thrombin analysis determined that coagulation activity was reduced relative to control at 12 min with the denucleation group (P < .05) and heparin group (P < .01) and at 18 min with all treatment groups (P < .01). The similarity of results among the techniques increases confidence that each measurement accurately predicts in vitro thrombogenicity.

Awards and Prizes↗

Nitric oxide synthase inhibition and extracellular glutamate concentration after cerebral ischemia/reperfusion.

BACKGROUND AND PURPOSE: Transient cerebral ischemia in rats results in selective loss of neuronal viability, eg, hippocampal CA1 neurons. The neurochemical variables responsible for this selective vulnerability to ischemia/reperfusion (IR) appear to involve excitatory amino acids. In brain IR, excitatory amino acid toxicity may be modulated by endogenous nitric oxide (NO.) gas. To investigate NO. in global brain IR, we measured the effects of NO. synthase (NOS) inhibition on interstitial excitatory amino acids in rats. Changes in postischemic cerebral blood flow and blood-brain barrier function also were evaluated. METHODS: Forebrain ischemia was produced by systemic hypotension and occlusion of both carotid arteries for 15 minutes. Blood flow was restored for 60 minutes by unclamping the carotids and reinfusing with blood. A microdialysis probe was placed into the cortex and hippocampus using a stereotaxic device. Interstitial glutamate concentration was measured during IR with high-performance liquid chromatography. A competitive NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), was given intraperitoneally 30 minutes before ischemia in doses of 1, 4, and 20 mg/kg. Changes in cerebral blood flow and blood-brain barrier during IR were determined using laser-Doppler flowmetry and microdialysis with sodium fluorescein. RESULTS: Glutamate in the dialysate during IR increased transiently 10-fold and returned to baseline levels by 30 minutes of reperfusion. Animals treated with L-NAME 30 minutes before ischemia also showed increases in glutamate concentration during ischemia, but glutamate remained elevated during reperfusion. The increase in glutamate concentration during reperfusion caused by L-NAME was prevented by L-arginine. The administration of L-arginine and L-NAME together decreased extracellular glutamate concentration during ischemia. Cerebral blood flow decreased to about 5% of baseline values during ischemia but increased approximately fourfold relative to control values on reperfusion. The hyperemic responses after ischemia were not different between IR groups treated with or without L-NAME. Brain ischemia increased the permeability of the blood-brain barrier to fluorescein; however, this change was attenuated by L-NAME administration at 20 mg/kg. CONCLUSIONS: NOS inhibition did not attenuate extracellular glutamate accumulation during ischemia and increased its concentration on reperfusion. The elevated glutamate concentration after IR in L-NAME-treated rats did not appear to be due to either a decrease in cerebral blood flow response after ischemia or increases in local blood-brain barrier permeability. For the most part, the blood-brain barrier was spared in the immediate postischemic period by L-NAME treatment. These data suggest that NO. production may oppose synaptic excitatory amino acid accumulation and presumably excitotoxicity during IR.

Amino Acid Oxidoreductases↗

Inhibition of nitric oxide synthase on brain oxygenation in anesthetized rats exposed to hyperbaric oxygen.

Nitric oxide (NO) production is involved in the development of oxygen toxicity of the central nervous system (CNS) since inhibition of nitric oxide synthase (NOS) significantly protects animals from hyperbaric oxygen (HBO)-mediated convulsions. One potential mechanism for this protection is that NOS inhibition decreases cerebral O2 delivery thereby limiting the PO2 of brain tissues during hyperoxia. To investigate this hypothesis, anesthetized rats were exposed to 7, 100, and 7% O2 under 3 atm abs for 15-min periods. Cortical blood flow (CBF) and O2 tension were measured with a laser-Doppler flowprobe and an O2 electrode, respectively, with and without pretreatment with the NOS doppler, N omega-nitro-L-arginine methyl ester (L-NAME). We found that HBO exposure significantly increased the brain O2 tension whereas changes in CBF were not significant. Compared with control rats, L-NAME administration did not change either brain O2 tension or CBF during the period of the experiment. We conclude that the effects of L-NAME on cortical oxygenation and CBF during HBO exposure in rats do not seem to provide a physiologic explanation for protection from CNS O2 toxicity by the drug.

Anesthesia↗

Blood-flow mapping of oral tissues by laser Doppler flowmetry.

The purpose of this study was to develop a rapid protocol for noninvasive quantification of blood flow at intraoral sites by laser Doppler flowmetry. For each flow measurement, the lowest flow observed over a 30-s period was recorded. Three flow readings at each site were averaged and recorded. Forehead and dorsal right-hand blood flows measured agreed with previous laser Doppler flow measurements. Flows measured with a hand-held probe were not different from flows measured with a stent-stabilized probe on the attached gingiva and an adhesive-stabilized probe on the dorsal hand. High flows were found in the tongue, buccal mucosa, and buccal vestibule. Medium flows were found in the attached gingivae. The lowest flows were found in the teeth. These results indicate that flow in intraoral tissues varies by site and can be noninvasively quantified with the laser Doppler flowmeter when a rigorous measurement protocol is used.

Adult↗

Differences in leucocyte-endothelium interactions between normal and adenocarcinoma bearing tissues in response to radiation.

Previously, we demonstrated that the interaction between leucocytes and endothelial cells in tumour tissues is greatly diminished compared with normal tissues under several induced inflammatory conditions. Radiation has been reported to cause release of inflammatory mediators and to promote neutrophil adhesions to cultured endothelial monolayers. In this study, we tested the hypothesis that radiation would cause increased leucocyte rolling and adhesion in both tumour and normal tissues. We examined these two parameters in response to 6 Gy of gamma-radiation in mammary adenocarcinomas implanted into rat skinfold window chambers as well as normal (i.e. non-tumour-bearing) preparations. Leucocyte rolling and adhesion were measured in terms of flux of rolling leucocytes (F(rolling)) and density of adhering leucocytes (D(adhering)) in microvessels. F(rolling) and D(adhering) were measured in two groups of preparations: irradiated and control. In normal preparations, F(rolling) and D(adhering) were both increased significantly by radiation. In contrast, in adenocarcinoma-bearing preparations, F(rolling) and D(adhering) were either unchanged (in the tumour centre) or reduced (in tumour periphery and the normal tissue surrounding the tumour) by radiation. Radiation did not cause changes in haemodynamics in these preparations, thus the observed changes in leucocyte rolling and adhesion could not be accounted for by haemodynamic factors. These results indicate that: (1) in normal preparations, radiation could cause inflammation as manifested by increased leucocyte rolling and adhesion; and (2) in tumour-bearing preparations, radiation caused changes in the vascular surface properties such that they became less adhesive to leucocytes. Such differences in radiation response may have important implications for radiation therapy and provide new insights into the unique features of tumours.

Adenocarcinoma↗

Evaluation of the thrombogenecity of microvascular prosthesis by in vivo microscopy.

Expanded polytetrafluoroethylene(ePTFE) grafts 4mm long and 1mm in diameter were implanted into the iliac artery of 100-150g male rats using standard microvascular technique. Prior to clamp removal, the cremaster muscle was isolated as an island flap based on the iliac artery and observed using intravital fluorescence microscopy. Fields which contained a bifurcation of a first order arteriole(80-100 microns diameter) into second order arteriole(50-80 microns) were chosen for observation. Platelets were labeled in vivo with acridine red to visualize and quantify the aggregates. Images of microemboli were counted manually and the area was measured by computerized planimetry. Six control grafts were implanted with no further processing, six were irrigated with heparin, and six were coated with tridodecylmethylammonium chloride(TDMAC) and heparin. Most thrombi appeared within the first five minutes after implantation in all groups. The total number of emboli observed in the control group was 91 pr animal, in the heparin irrigation group it was 84, and in the TDMAC-heparin group it was 22. The total thrombus area observed per animal was 137,660 +/- 29,467 microns 2 in the control group, 79,040 +/- 10,893 microns 2 in the heparin irrigation group, and 17,498 +/- 6,059 microns 2 in the TDMAC-heparin group (p < .01 vs control or heparin irrigation group). With this results we could find that heparin irrigation and TDMAC-heparin coating appear to reduce the number, size, and total amount of microemboli generated by ePTFE graft implantation and apparent thromboresistant property of TDMAC-heparin coating may have widespread application in many clinical and research areas and this experimental model can be used for evaluation of other graft matrials.

Animals↗

Analysis of oxygen transport to tumor tissue by microvascular networks.

We present theoretical simulations of oxygen delivery to tumor tissues by networks of microvessels, based on in vivo observations of vascular geometry and blood flow in the tumor microcirculation. The aim of these studies is to investigate the impact of vascular geometry on the occurrence of tissue hypoxia. The observations were made in the tissue (thickness 200 microns) contained between two glass plates in a dorsal skin flap preparation in the rat. Mammary adenocarcinomas (R3230 AC) were introduced and allowed to grow, and networks of microvessels in the tumors were mapped, providing data on length, geometric orientation, diameter and blood velocity in each segment. Based on these data, simulations were made of a 1 mm x 1 mm region containing five unbranched vascular segments and a 0.25 mm x 0.35 mm region containing 22 segments. Generally, vessels were assumed to lie in the plane midway between the glass plates, at 100 microns depth. Flow rates in the vessels were based on measured velocities and diameters. The assumed rate of oxygen consumption in the tissue was varied over a range of values. Using a Green's function method, partial pressure of oxygen (PO2) was computed at each point in the tissue region. As oxygen consumption is increased, tissue PO2 falls, with hypoxia first appearing at points relatively distant from the nearest blood vessel. The width of the well-oxygenated region is comparable to that predicted by simpler analyses. Cumulative frequency distributions of tissue PO2 were compared with predictions of a Krogh-type model with the same vascular densities, and it was found that the latter approach, which assumes a uniform spacing of vessels, may underestimate the extent of the hypoxic tissue. Our estimates of the maximum consumption rate that can be sustained without tissue hypoxia were substantially lower than those obtained from the Krogh-type model. We conclude that the heterogeneous structure of tumor microcirculation can have a substantial effect on the occurrence of hypoxic micro-regions.

Adenocarcinoma↗

A comparison of tumor and normal tissue microvascular hematocrits and red cell fluxes in a rat window chamber model.

This laboratory has previously used a window chamber model to measure red blood cell velocity in mammary tumors and normal granulation tissues of the F-344 rat. Because red cell flux and hematocrit more accurately reflect the oxygen carrying potential of blood, we used this model to measure these parameters. Red blood cells were labelled with fluorescein isothiocyanate, and 0.2 ml. packed cells were injected intravenously into rats bearing an 8 to 10 day old R-3230 mammary carcinoma. beta-phycoerythrin (0.15 mg.) was also injected and served as a plasma dye to outline the blood vessels. A sample of peripheral blood was then taken and analyzed by flow cytometry to determine the labeled fraction of red blood cells. Flowing tumor and normal tissue vessels were recorded onto a VCR, and these video images were used to determine vascular length and diameter, RBC flux and velocity, and hematocrit. Median vessel diameter and loge (red blood cell flux) were significantly greater in tumors than in normal tissues (p = 0.007 and p < 0.025, respectively). After controlling for these variables, the median tumor hematocrit of 19% was not significantly greater than the median normal tissue hematocrit of 15%. This technique provides a nontoxic and reproducible method that is now being used to assist in the in vivo definition of tumor oxygenation.

Animals↗