Proapoptotic effects of novel thiazole derivative on human glioma cells.

The aim of the present study was to investigate the antiproliferative and proapoptotic actions of N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide derivative (compound 5) in glioma cells in comparison with the actions of temozolomide (TMZ) and doxorubicin (Dox), used as positive controls. The antiproliferative activity of the compound 5, TMZ, and Dox on human glioblastoma U251 and human glioblastoma multiform T98G cells was measured using the MTT test. Western blot analysis, fluorescent microscopy, agarose gel retardation assay, flow cytometric analysis, and the DNA comet assay under alkaline conditions were carried out to study the effect of compound 5 on U251 cells. This compound showed ~20 times higher cytotoxicity toward U251 and T98G cells compared with the effects of TMZ and approximately two times higher activity than that of the Dox. Compound 5 induced apoptosis in U251 cells by PARP1 and caspase 3 cleavage mechanisms, also inducing an increase in the level of Bax and Bim proapoptotic proteins and a decrease in the level of phosho-ERK1/2 kinase. The cytotoxicity of compound 5 was associated with an increase in the production of the hydrogen peroxide and the formation of DNA single-strand breaks. This compound 5 did not intercalate into a DNA molecule. Thus, the novel thiazole derivative (compound 5) proved to be a potential antiglioma drug that showed much higher cytotoxic action on human glioma cells compared with the effects of TMZ and Dox. Its cytotoxicity is associated with apoptosis induction, production of the reactive oxygen species, and formation of DNA single-strand breaks without significant DNA intercalation.

The Role of Sodium in Diabetic Cardiomyopathy.

Cardiovascular complications are the major cause of mortality and morbidity in diabetic patients. The changes in myocardial structure and function associated with diabetes are collectively called diabetic cardiomyopathy. Numerous molecular mechanisms have been proposed that could contribute to the development of diabetic cardiomyopathy and have been studied in various animal models of type 1 or type 2 diabetes. The current review focuses on the role of sodium (Na+) in diabetic cardiomyopathy and provides unique data on the linkage between Na+ flux and energy metabolism, studied with non-invasive 23Na, and 31P-NMR spectroscopy, polarography, and mass spectroscopy. 23Na NMR studies allow determination of the intracellular and extracellular Na+ pools by splitting the total Na+ peak into two resonances after the addition of a shift reagent to the perfusate. Using this technology, we found that intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control possibly due to combined changes in the activity of Na+-K+ pump, Na+/H+ exchanger 1 (NHE1) and Na+-glucose cotransporter. We hypothesized that the increase in Na+ activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to a loss of intramitochondrial Ca2+, with a subsequent alteration in mitochondrial bioenergetics and function. Using isolated mitochondria, we showed that the addition of Na+ (1-10 mM) led to a dose-dependent decrease in oxidative phosphorylation and that this effect was reversed by providing extramitochondrial Ca2+ or by inhibiting the mitochondrial Na+/Ca2+ exchanger with diltiazem. Similar experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels and that this effect was stronger in diabetic rats. These data suggest that in diabetic cardiomyocytes, increased Na+ leads to abnormalities in oxidative phosphorylation and a subsequent decrease in ATP levels. In support of these data, using 31P-NMR, we showed that the baseline ß-ATP and phosphocreatine (PCr) were lower in diabetic cardiomyocytes than in control, suggesting that diabetic cardiomyocytes have depressed bioenergetic function. Thus, both altered intracellular Na+ levels and bioenergetics and their interactions may significantly contribute to the pathology of diabetic cardiomyopathy.

AMP promotes oxygen consumption and ATP synthesis in heart mitochondria through the adenylate kinase reaction: an NMR spectroscopy and polarography study.

Adenylate kinase plays an important role in cellular energy homeostasis by catalysing the interconversion of adenine nucleotides. The goal of present study was to evaluate the contribution of the adenylate kinase reaction to oxidative ATP synthesis by direct measurements of ATP using (31) P NMR spectroscopy. Results show that AMP can stimulate ATP synthesis in the presence or absence of ADP. In particular, addition of 1 mM AMP to the 0.6 mM ADP superfusion system of isolated superfused mitochondria (contained and maintained in agarose beads) led to a 25% increase in ATP synthesis as measured by the increase in ßATP signal. More importantly, we show that AMP can support ATP synthesis in the absence of ADP, demonstrated as follows. Superfusion of mitochondria without ADP led to the disappearance of ATP γ, α and ß signals and the increase of Pi . Addition of AMP to the medium restored the production of ATP, as demonstrated by the reappearance of γ, α and ß ATP signals, in conjunction with a decrease in Pi , which is being used for ATP synthesis. Polarographic studies showed Mg(2+) dependence of this process, confirming the specificity of the adenylate kinase reaction. Furthermore, data obtained from this study demonstrate, for the first time, that different aspects of the adenylate kinase reaction can be evaluated with (31) P NMR spectroscopy. SIGNIFICANCE OF RESEARCH PARAGRAPH: The data generated in the present study indicate that (31) P NMR spectroscopy can effectively be used to study the adenylate kinase reaction under a variety of conditions. This is important because understanding of adenylate kinase function and/or malfunction is essential to understanding its role in health and disease. The data obtained with (31) P NMR were confirmed by polarographic studies, which further strengthens the robustness of the NMR findings. In summary, (31) P NMR spectroscopy provides a sensitive tool to study adenylate kinase activity in different physiological and pathophysiological conditions, including but not exclusive of, cancer, ischemic injury, hemolytic anemia and neurological problems such as sensorineural deafness.

Variability of apoptosis and response in N1-S1 rodent hepatomas to benzamide riboside and correlation to early changes in water apparent diffusion coefficient and sodium MR imaging.

PURPOSE: This pilot trial assesses variability of apoptosis and response 1 day after hepatic intraarterial (IA) benzamide riboside (BR) in rodent hepatomas and its correlation to water apparent diffusion coefficient (ADC) and single-quantum (SQ) and triple-quantum-filtered (TQF) sodium-23 ((23)Na) magnetic resonance (MR) imaging. MATERIALS AND METHODS: Sprague-Dawley rats (n = 8) were inoculated with 10(6) N1-S1 cells. IA BR (20 mg/kg) was infused after 14 days. Animals were killed 1 day (n = 4) or 21 days (n = 4) after therapy. Imaging was performed 1 day before and after treatment. Volume was assessed over 2 weeks. Percentage apoptosis was counted from terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained slides at 400×magnification. Kruskal-Wallis tests were used to compare apoptosis, and Wilcoxon signed-rank tests were used to compare MR signal intensity (SI). RESULTS: Apoptosis was marginally greater in tumor than in nontumor (6.7% vs 1.3%; P = .08), varying from 2% to 10%. Before treatment, MR SI was greater in tumor than in nontumor (ADC, 1.18 vs 0.76 [P = .0078]; SQ, 1.20 vs 1.04 [P = .03]; TQF, 0.55 vs 0.34 [P = .03]). After treatment, tumors increased in volume (0.62 vs 0.33; P = .016) variably over 2 weeks. MR SI remained greater in tumor than in nontumor (ADC, 1.20 vs 0.77 [P = .0078]; SQ, 1.76 vs 1.15 [P = .016]; TQF, 0.84 vs 0.49 [P = .03]). SQ and TQF SI increased by 47% (P = .016) and 53% (P = .016) in tumors, whereas ADC did not change. CONCLUSIONS: Apoptosis was marginal and varied from 2% to 10%. Water ADC, SQ, and TQF MR imaging distinguished tumor from nontumor. Changes in water ADC and sodium MR imaging correlated to apoptosis and volume in select cases, but additional animals are needed to validate this trend against tumor growth.

Tumor response and apoptosis of N1-S1 rodent hepatomas in response to intra-arterial and intravenous benzamide riboside.

PURPOSE: Benzamide riboside (BR) induces tumor apoptosis in multiple cell lines and animals. This pilot study compares apoptosis and tumor response in rat hepatomas treated with hepatic arterial BR (IA) or intravenous (IV) BR. METHODS: A total of 10(6) N1-S1 cells were placed in the left hepatic lobes of 15 Sprague-Dawley rats. After 2 weeks, BR (20 mg/kg) was infused IA (n=5) or IV (n=5). One animal in each group was excluded for technical factors, which prevented a full dose administration (1 IA and 1 IV). Five rats received saline (3 IA and 2 IV). Animals were killed after 3 weeks. Tumor volumes after IA and IV treatments were analyzed by Wilcoxon rank sum test. The percentage of tumor and normal liver apoptosis was counted by using 10 fields of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-stained slides at 40× magnification. The percentage of apoptosis was compared between IV and IA administrations and with saline sham-treated rats by the Wilcoxon rank sum test. RESULTS: Tumors were smaller after IA treatment, but this did not reach statistical significance (0.14 IA vs. 0.57 IV; P=0.138). There was much variability in percentage of apoptosis and no significant difference between IA and IV BR (44.49 vs. 1.52%; P=0.18); IA BR and saline (44.49 vs. 33.83%; P=0.66); or IV BR and saline (1.52 vs. 193%; P=0.18). CONCLUSIONS: Although differences in tumor volumes did not reach statistical significance, there was a trend toward smaller tumors after IA BR than IV BR in this small pilot study. Comparisons of these treatment methods will require a larger sample size and repeat experimentation.

Effect of implantation site and growth of hepatocellular carcinoma on apparent diffusion coefficient of water and sodium MRI.

Hepatocellular carcinoma (HCC) and liver metastases are an increasing problem worldwide. Non-invasive methods for the early detection of HCC and understanding of the tumor growth mechanisms are highly desirable. Both the diffusion-weighted (1)H (DWI) and (23)Na MRI reflect alterations in tissue compartment volumes in tumors, as well as physiological and metabolic transformation in cells. Effects of untreated growth on apparent diffusion coefficient of water (ADC), single quantum (SQ) and triple quantum-filtered (TQF) (23)Na MRI were compared in intrahepatically and subcutaneously implanted HCCs in rats. Animals were examined weekly for 4 weeks after injection of N1S1 cells. ADC of intrahepatic HCC was 1.5-times higher compared to the nearby liver tissue, and with growth, the ADC did not increase. ADC of subcutaneous HCC was lower compared to intrahepatic HCC and it increased with growth. Untreated growth of both intrahepatic and subcutaneous HCCs was associated with an increase in SQ and TQF (23)Na signal intensity suggesting an increase in tissue Na(+) and intracellular Na(+) (Na(+)(i)), respectively, most likely due to an increase in relative extracellular space and Na(+)(i) concentration as a result of changes in tissue structure and cellular metabolism. Thus, SQ and TQF (23)Na MRI may be complementary to diffusion imaging in areas susceptible to motion for characterizing hepatic tumors and for other applications, such as, predicting and monitoring therapy response.

Predicting response to benzamide riboside chemotherapy in hepatocellular carcinoma using apparent diffusion coefficient of water.

AIM: To monitor the effects of the apoptotic agent benzamide riboside (BR) on tumor volume and water apparent diffusion coefficient (ADC) in rat hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Water ADC of the tumors and nearby liver tissue was measured using diffusion-weighted 1H MRI (DWI). The two groups of BR-treated animals, which differed in their sensitivity to the treatment, were identified as responsive (RBR) and non-responsive (NRBR). RESULTS: Tumor growth in the RBR group was arrested and the mean tumor volume in this group was 1/6th and 1/16th compared to that of the NRBR group on days 7 and 14 after treatment, respectively. Water ADC of HCC was higher than in nearby normal liver tissue. Before BR treatment, the mean water ADC was significantly higher in the RBR group compared to the NRBR group. BR therapy did not change the water ADC value regardless of tumor sensitivity. CONCLUSION: Although the water ADC did not change after chemotherapy by BR, DWI has great potential for detecting and predicting response to chemotherapy in HCC.

Controlled radio-frequency hyperthermia using an MR scanner and simultaneous monitoring of temperature and therapy response by (1)H, (23)Na and (31)P magnetic resonance spectroscopy in subcutaneously implanted 9L-gliosarcoma.

A magnetic resonance (MR) technique is developed to produce controlled radio-frequency (RF) hyperthermia (HT) in subcutaneously-implanted 9L-gliosarcoma in Fisher rats using an MR scanner and its components; the scanner is also simultaneously used to monitor the tumour temperature and the metabolic response of the tumour to the therapy. The method uses the (1)H chemical shift of thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid (TmDOTA(-)) to monitor temperature. The desired HT temperature is achieved and maintained using a feedback loop mechanism that uses a proportional-integral-derivative controller. The RF HT technique was able to heat the tumour from 33 degrees to 45 degrees C in approximately 10 min and was able to maintain the tumour temperature within +/-0.2 degrees C of the target temperature (45 degrees C). Simultaneous monitoring of the metabolic changes with RF HT showed increases in total tissue and intracellular Na(+) as measured by single-quantum and triple-quantum filtered (23)Na MR spectroscopy (MRS), respectively, and decreases in intra- and extracellular pH and cellular bioenergetics as measured by (31)P MRS. Monitoring of metabolic response in addition to the tumour temperature measurements may serve as a more reliable and early indicator of therapy response. In addition, such measurements during HT treatment will enhance our understanding of the tumour response mechanisms during HT, which may prove valuable in designing methods to improve therapeutic efficiency.

Monitoring intrahepatic hepatocellular carcinoma growth by 23Na-MRI and 1H-diffusion weighted imaging / 中华放射学杂志

Objective To monitor the structural and metabolic transformation of growing intrahepatic hepatocellular carcinoma (HCC) by non-invasive 23Na-MRI and 1H-DWI. Methods Each animal was examined weekly for4 weeks after injection of 1 × 106 N1S1 cells into the left liver lobe. MR images were acquired with a Varian MR system. The effects of untreated growth on water apparent diffusion coefficient (ADC), total tissue Na+ and intracellular Na+ were monitored in rat HCCs using 1H-DWI,single-quantum ( SQ ) 23 Na and triple-quantum-filtered (TQF) 23 Na-MRI. Histological analysis of HCC tissues was performed. Relative extracellular space (ECS), cell numbers, and the cell to nucleus area ratio were calculated. Statistical analysis of the data was performed by ANOVA and post-hoc multiple comparison.Results The doubling time of the tumor growth was 3.9 days. During the four weeks in tumor growing, the ratio of water ADC to nearby liver was always 1.4 to 1.5. The HCC growth was associated with an increase in both total tissue and intracellular 23Na signal intensity, and the changes in Nai+ were more profound than in Nat+. The ratio of the tumor SQ, TQF 23Na signal intensity to the nearby liver at four time points(7,14,21 and28 d) was 1.05 ±0.20,1.41 ±0.32,1.50±0.45,1.62±0.50(F=2.97,P＜0.05);1.32±0.11,1.54 ± 0.18,2.38 ± 0.22,2.39 ± 0.16 ( F = 11.18, P ＜ 0.01 ), respectively. Statistical analysis of the histological slices showed that the mean of relative ECS in healthy liver was significantly lower compared to both ECS spaces in HCC viable cells and inflammation/necrosis areas. The number of viable and inflamed/necrotic cells was statistically higher compared to nearby liver tissue. However, the increase in "cellularity" was caused mostly by shrinkage of cellular cytoplasm when the ratio of cell to nucleus areas decreased from 4.0 ± 0.3 ( normal liver) to 1.6 ± 0.1 ( viable HCC cells) ( t = 20.08,P ＜ 0.05). Conclusions The water ADC and SQ23 Na-MRI reflect ECS, total tissue Na+ changes and thus reflect mostly the compartmental alterations in tumor tissue, while TQF 23Na-MRI reflects the intracellular Na+ and thus physiological,metabolic transformation in HCC cells.

Early monitoring of acute tubular necrosis in the rat kidney by 23Na-MRI.

Reabsorption of water and other molecules is dependent on the corticomedullary sodium concentration gradient in the kidney. During the early course of acute tubular necrosis (ATN), this gradient is altered. Therefore, 23Na magnetic resonance imaging (MRI) was used to study the alterations in renal sodium distribution in the rat kidney during ischemia and reperfusion (IR) injury, which induces ATN. In-magnet ischemia was induced for 0 (control), 10, 20, 30 or 50 min in Wistar rats. 23Na images were collected every 10 min during baseline, ischemia, and 60-min reperfusion periods. T1 and T2 relaxation times were measured by both 23Na-MRI and -MRS on a separate cohort of animals during ischemia and reperfusion for correction of relaxation-related tissue sodium concentration (TSC). A marked decrease was observed in the medulla and cortex 23Na-MRI signal intensity (SI) during the early evolution of ATN caused by IR injury, with the sodium reabsorption function of the kidney being irreversibly damaged after 50 min of ischemia. Sodium relaxation time characteristics were similar in the medulla and cortex of normal kidney, but significantly decreased with IR. The changes in relaxation times in both compartments were identical; thus the medulla-to-cortex sodium SI ratio represents the TSC ratio of both compartments. The extent of IR damage observed with histological examination correlated with the 23Na-MRI data. 23Na-MRI has great potential for noninvasive, clinical diagnosis of evolving ATN in the setup of acute renal failure and in differentiating ATN from other causes of renal failure where tubular function is maintained.

Absolute temperature MR imaging with thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetic acid (TmDOTMA-).

MR thermometry based on the water (1)H signal provides high temporal and spatial resolution, but it has low temperature sensitivity (approximately 0.01 ppm/degrees C) and requires monitoring of another weaker signal for absolute temperature measurements. The use of the paramagnetic lanthanide complex, thulium 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetate (TmDOTMA(-)), which is approximately 60 times more sensitive to temperature than the water (1)H signal, is advanced to image absolute temperatures in vivo using water signal as a reference. The temperature imaging technique was developed using gradient echo and asymmetric spin echo imaging sequences on 9.4 Tesla (T) horizontal and vertical MR scanners. A comparison of regional temperatures measured with TmDOTMA(-) and fiber-optic probes showed that the accuracy of imaging temperature is <0.3 degrees C. The temperature imaging technique was found to be insensitive to inhomogeneities in the main magnetic field. The feasibility of imaging temperature of intact rats at approximately 1.4 mmol/kg dose with approximately 1-mm spatial resolution in only 3 min is demonstrated. TmDOTMA(-) should prove useful for imaging absolute temperatures in deep-seated organs in numerous biomedical applications.

Fat and water 1H MRI to investigate effects of leptin in obese mice.

Leptin is known to be associated with regulation of body weight and fat content. The effects of exogenous leptin on abdominal visceral (VS) and subcutaneous (SC) fat volume and hepatic fat-to-water ratio in leptin-deficient obese mice were investigated by (1)H magnetic resonance imaging (MRI). Chemical shift-selected fat and water (1)H MRI of control and leptin-treated mice were obtained 1 day before treatment and after 7 days of treatment (0.3 mg/kg/day). Hepatic fat-to-water ratio and VS fat volume decreased significantly with treatment, whereas SC fat volume did not change. Noninvasive measurement of fat and water content in different body regions using MRI should prove useful for evaluating new drugs for the treatment of obesity and other metabolic disorders.

Evaluation of extra- and intracellular apparent diffusion coefficient of sodium in rat skeletal muscle: effects of prolonged ischemia.

The mechanism of water and sodium apparent diffusion coefficient (ADC) changes in rat skeletal muscle during global ischemia was examined by in vivo 1H and 23Na magnetic resonance spectroscopy (MRS). The ADCs of Na+ and water are expected to have similar characteristics because sodium is present as an aqua-cation in tissue. The shift reagent, TmDOTP5(-), was used to separate intra- and extracellular sodium (Na+i and Na+e, respectively) signals. Water, total tissue sodium (Na+t), Na+i, and Na+e ADCs were measured before and 1, 2, 3, and 4 hr after ischemia. Contrary to the general perception, Na+i and Na+e ADCs were identical before ischemia. Thus, ischemia-induced changes in Na+e ADC cannot be explained by a simple change in the size of relative intracellular or extracellular space. Na+t and Na+e ADCs decreased after 2-4 hr of ischemia, while water and Na+i ADC remained unchanged. The correlation between Na+t and Na+e ADCs was observed because of high Na+e concentration. Similarly, the correlation between water and Na+i ADCs was observed because cells occupy 80% of the tissue space in the skeletal muscle. Ischemia also caused an increase in the Na+i and an equal decrease in Na+e signal intensity due to cessation of Na+/K+-ATPase function.

Monitoring chemotherapeutic response in RIF-1 tumors by single-quantum and triple-quantum-filtered (23)Na MRI, (1)H diffusion-weighted MRI and PET imaging.

The effects of 5-fluorouracil (5FU, 150 mg/kg, ip) on subcutaneously implanted radiation-induced fibrosarcoma (RIF-1) tumors were monitored by in vivo (1)H MRI to evaluate the water apparent diffusion coefficient (ADC), by single-quantum (SQ) and triple-quantum-filtered (TQF) (23)Na MRI to evaluate compartmental Na(+) content and by positron emission tomography (PET) to evaluate 2-[(18)F]fluoro-2-deoxy-d-glucose (FDG) uptake in the tumor. The MRI experiments were performed on untreated control and treated mice once before and then daily for 3 days after treatment. The PET experiments were performed on separate groups of age- and tumor-volume-matched animals once before and then 3 days after treatment. Tumor volumes significantly decreased in treated animals 2 and 3 days posttreatment. At the same time points, in vivo MRI measurements showed an increase in both total tissue SQ (23)Na signal intensity (SI) and water ADC in treated tumors while control tumors showed no change in these parameters. TQF (23)Na SI and FDG uptake were significantly lower in treated tumors compared with control tumors 3 days after 5FU treatment. The correlated increases in total tissue (23)Na SI and water ADC following chemotherapy reflect an increase in extracellular space, while the lower TQF (23)Na SI and FDG uptake in treated tumors compared with control tumors suggest a shift in tumor metabolism from glycolysis to oxidation and/or a decrease in cell density.

Predicting and monitoring response to chemotherapy by 1,3-bis(2-chloroethyl)-1-nitrosourea in subcutaneously implanted 9L glioma using the apparent diffusion coefficient of water and 23Na MRI.

PURPOSE: To examine the effects of the alkylating anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on (23)Na MRI and the water apparent diffusion coefficient (ADC) in subcutaneously- (sc-) implanted 9L glioma in rats. MATERIALS AND METHODS: (23)Na MRI and (1)H water ADC measurements were performed on sham-treated control (N = 6) and BCNU-treated (N = 15) Fisher rats one day before BCNU injection and then one, three, and five days after BCNU injection. RESULTS: The BCNU-treated tumors were divided into BCNU-responsive (R(BCNU)) and BCNU-nonresponsive (NR(BCNU)) groups depending on the tumor volume changes that occurred after therapy. The pretreatment (23)Na MRI signal intensity (SI) and water ADC values were higher in R(BCNU) tumors compared to NR(BCNU) tumors. (23)Na MRI SI and water ADC increased with tumor growth in control and NR(BCNU) groups, but these changes were interrupted by BCNU therapy in R(BCNU) group. CONCLUSION: (23)Na MRI and water ADC measurements may be useful for predicting and monitoring response to chemotherapy in some tumors. However, the changes that occurred in (23)Na MRI SI and water ADC in sc-implanted 9L tumors are in contrast to previously published results for BCNU therapy of orthotopic 9L tumors. This may have important implications for monitoring therapy response in tumors.

Non-invasive temperature imaging with thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetic acid (TmDOTMA-).

Non-invasive thermometry using hyperfine-shifted MR signals from paramagnetic lanthanide complexes has attracted attention recently because the chemical shifts of these complexes are many times more sensitive to temperature than the water 1H signal. Among all the lanthanide complexes examined thus far, thulium tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTMA-) appears to be the most suitable for MR thermometry. In this paper, the feasibility of imaging the methyl 1H signal from TmDOTMA- using a frequency-selective radiofrequency excitation pulse and chemical shift-selective (CHESS) water suppression is demonstrated. A temperature imaging method using a phase-sensitive spin-echo imaging sequence was validated in phantom experiments. A comparison of regional temperature changes measured with fiber-optic probes and the temperatures calculated from the phase shift near each probe showed that the accuracy of imaging the temperature with TmDOTMA- is at least 0.1-0.2 degrees C. The feasibility of imaging temperature changes in an intact rat at 0.5-0.6 mmol/kg dose in only a few minutes is demonstrated. Similar to commonly used MRI contrast agents, the lanthanide complex does not cross the blood-brain barrier. TmDOTMA- may prove useful for temperature imaging in many biomedical applications but further studies relating to acceptable dose and signal-to-noise ratio are necessary before clinical applications.

Application of 23Na MRI to monitor chemotherapeutic response in RIF-1 tumors.

Effects of an alkylating anticancer drug, cyclophosphamide (Cp), on 23Na signal intensity (23Na SI) and water apparent diffusion coefficient (ADC) were examined in subcutaneously-implanted radiation-induced fibrosarcoma (RIF-1) tumors by 23Na and 1H magnetic resonance imaging (MRI). MRI experiments were performed on untreated control (n = 5) and Cp-treated (n = 6) C3H mice, once before Cp injection (300 mg/kg) then daily for 3 days after treatment. Tumor volumes were significantly lower in treated animals 2 and 3 days posttreatment. At the same time points, in vivo MRI experiments showed an increase in both 23Na SI and water ADC in treated tumors, whereas control tumors did not show any significant changes. The correlation between 23Na SI and water ADC changes was dramatically increased in the Cp-treated group, suggesting that the observed increases in 23Na SI and water ADC were caused by the same mechanism. Histologic sections showed decreased cell density in the regions of increased 23Na and water ADC SI. Destructive chemical analysis showed that Cp treatment increased the relative extracellular space and tumor [Na+]. We conclude that the changes in water ADC and 23Na SI were largely due to an increase in extracellular space. 23Na MRI and 1H water ADC measurements may provide valuable noninvasive techniques for monitoring chemotherapeutic responses.

Noninvasive MR thermometry using paramagnetic lanthanide complexes of 1,4,7,10-tetraazacyclodoecane-alpha,alpha',alpha'',alpha'''-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA4-).

Noninvasive techniques to monitor temperature have numerous useful biomedical applications. However, MR thermometry techniques based on the chemical shift, relaxation rates, and molecular diffusion rate of the water 1H signal suffer from poor thermal resolution. The feasibility of MR thermometry based on the strong temperature dependence of the hyperfine-shifted 1H signal from the paramagnetic lanthanide complex thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA-) was recently demonstrated. The use of paramagnetic lanthanide complexes for MR thermometry can be further enhanced by improving the signal-to-noise ratio (SNR) of the observed signal. In this study, the use of lanthanide complexes of a methyl-substituted analog of DOTA4-, 1,4,7,10-tetramethyl 1,4,7,10-tetra azacyclodoecane-1,4,7,10-tetraacetic acetate (DOTMA4-) was evaluated. DOTMA4- complexes have 12 magnetically equivalent methyl protons, which provide an intense and sharper resonance compared to the corresponding DOTA- complexes. Experiments with paramagnetic Pr3+, Yb3+, Tb3+, Dy3+, and Tm3+ complexes of DOTMA4- showed that the Tm3+ complex is most favorable for MR thermometery because of the high temperature dependence of its chemical shift and its relatively narrow linewidth. The chemical shift of the methyl 1H signal from TmDOTMA- was approximately 60 times more sensitive to temperature than the water 1H shift and was insensitive to changes in concentration, pH, [Ca2+], or the presence of other ions and macromolecules. The application of TmDOTMA- for measuring temperature in a subcutaneously implanted tumor model was demonstrated. Lastly, the feasibility of obtaining 3D images from the methyl 1H resonance of TmDOTMA- was demonstrated in phantom and live animal experiments. Overall, TmDOTMA- appears to be a promising probe for MR thermometry in vivo.

Effects of temperature on intracellular sodium, pH and cellular energy status in RIF-1 tumor cells.

Most perfused tumor cell experiments are performed at 37 degrees C, the normal healthy body temperature. However, the temperature of subcutaneously implanted tumors in small animals is generally 29-33 degrees C when the rectal temperature of the animal is maintained at 37 degrees C. We have investigated the acute effects of increasing the temperature of perfused radiation-induced-fibrosarcoma (RIF-1) tumor cells from 33 to 37 degrees C (30 min) on intracellular sodium (Na(i)+) , intracellular pH (pH(i)), and bioenergetic status. Heating the cells by 4 degrees C produced a reversible increase in Na(i)+, slight acidification and no change in nucleotide triphosphate to inorganic phosphate ratio (NTP/P(i)) as measured by shift-reagent-aided (23)Na and (31)P NMR spectroscopy. In the presence of 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a potent and specific inhibitor of Na(+)/H(+) antiporter, the increase in Na(i)+ during the heating was completely abolished suggesting that the heat induced increase in Na(i)+ was caused by an increase in Na(+)/H(+) antiporter activity. However, the changes in pH(i) with the heating were identical with or without EIPA, indicating that pH(i) is controlled by other ion exchange mechanisms in addition to Na(+)/H(+) antiporter. NTP/P(i) was significantly higher in presence of EIPA for some time points during the heating suggesting that both NTP production and consumption rates may be altered during the heating. These results indicate that a slight increase in temperature from 33 to 37 degrees C induces significant changes in Na(+) physiology largely because of activation of Na(+)/H(+) antiporter but other ion exchange mechanisms are also involved in maintaining pH(i) in the RIF-1 tumor cells. Thus, care must be taken in choosing the temperature for perfused cell studies.

Influence of ischemic preconditioning on intracellular sodium, pH, and cellular energy status in isolated perfused heart.

The possible relationships between intracellular Na(+) (Na(i)(+)), bioenergetic status and intracellular pH (pH(i)) in the mechanism for ischemic preconditioning were studied using (23)Na and (31)P magnetic resonance spectroscopy in isolated Langendorff perfused rat heart. The ischemic preconditioning (three 5-min ischemic episodes followed by two 5-min and one 10-min period of reperfusion) prior to prolonged ischemia (20 min stop-flow) resulted in a decrease in ischemic acidosis and faster and complete recovery of cardiac function (ventricular developed pressure and heart rate) after 30 min of reperfusion. The response of Na(i) during ischemia in the preconditioned hearts was characterized by an increase in Na(i)(+) at the end of preconditioning and an accelerated decrease during the first few minutes of reperfusion. During post-ischemic reperfusion, bioenergetic parameters (PCr/P(i) and betaATP/P(i) ratios) were partly recovered without any significant difference between control and preconditioned hearts. The reduced acidosis during prolonged ischemia and the accelerated decrease in Na(i)(+) during reperfusion in the preconditioned hearts suggest activation of Na(+)/H(+) exchanger and other ion transport systems during preconditioning, which may protect the heart from intracellular acidosis during prolonged ischemia, and result in better recovery of mechanical function (LVDP and heart rate) during post-ischemic reperfusion.