Therapeutic Effects of Microbubbles Added to Combined High-Intensity Focused Ultrasound and Chemotherapy in a Pancreatic Cancer Xenograft Model

OBJECTIVE: To investigate whether high-intensity focused ultrasound (HIFU) combined with microbubbles enhances the therapeutic effects of chemotherapy. MATERIALS AND METHODS: A pancreatic cancer xenograft model was established using BALB/c nude mice and luciferase-expressing human pancreatic cancer cells. Mice were randomly assigned to five groups according to treatment: control (n = 10), gemcitabine alone (GEM; n = 12), HIFU with microbubbles (HIFU + MB, n = 11), combined HIFU and gemcitabine (HIGEM; n = 12), and HIGEM + MB (n = 13). After three weekly treatments, apoptosis rates were evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay in two mice per group. Tumor volume and bioluminescence were monitored using high-resolution 3D ultrasound imaging and in vivo bioluminescence imaging for eight weeks in the remaining mice. RESULTS: The HIGEM + MB group showed significantly higher apoptosis rates than the other groups (p < 0.05) and exhibited the slowest tumor growth. From week 5, the tumor-volume-ratio relative to the baseline tumor volume was significantly lower in the HIGEM + MB group than in the control, GEM, and HIFU + MB groups (p < 0.05). Despite visible distinction, the HIGEM and HIGEM + MB groups showed no significant differences. CONCLUSION: High-intensity focused ultrasound combined with microbubbles enhances the therapeutic effects of gemcitabine chemotherapy in a pancreatic cancer xenograft model.

Erratum: Correction of Notes. In vitro MRI and Characterization of Rat Mesenchymal Stem Cells Transduced with Ferritin as MR Reporter Gene

We found an error in our published article.

In vitro MRI and Characterization of Rat Mesenchymal Stem Cells Transduced with Ferritin as MR Reporter Gene

PURPOSE: This study was performed to evaluate the characteristics of rat mesenchymal stem cells (RMSCs) transduced with human ferritin gene and investigate in vitro MRI detectability of ferritin-transduced RMSCs. MATERIALS AND METHODS: The RMSCs expressing both myc-tagged human ferritin heavy chain subunit (myc-FTH) and green fluorescence protein (GFP) were transduced with lentiviurs. Transduced cells were sorted by GFP expression using a fluorescence-activated cell sorter. Myc-FTH and GFP expression in transduced cells were detected by immunofluorescence staining. The cell proliferative ability and viability were assessed by MTT assay. The RMSC surface markers (CD29+/CD45-) were analyzed by flow cytometry. The intracellular iron amount was measured spectrophotometically and the presence of ferritin-iron accumulation was detected by Prussian blue staining. In vitro magnetic resonance imaging (MRI) study of cell phantoms was done on 9.4 T MR scanner to evaluate the feasibility of imaging the ferritin-transduced RMSCs. RESULTS: The myc-FTH and GFP genes were stably transduced into RMSCs. No significant differences were observed in terms of biologic properties in transduced RMSCs compared with non-transduced RMSCs. Ferritin-transduced RMSCs exhibited increased iron accumulation ability and showed significantly lower T2 relaxation time than non-transduced RMSCs. CONCLUSION: Ferritin gene as MR reporter gene could be used for non-invasive tracking and visualization of therapeutic mesenchymal stem cells by MRI.

Evaluation of Porcine Pancreatic Islets Transplanted in the Kidney Capsules of Diabetic Mice Using a Clinically Approved Superparamagnetic Iron Oxide (SPIO) and a 1.5T MR Scanner

OBJECTIVE: To evaluate transplanted porcine pancreatic islets in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner. MATERIALS AND METHODS: Various numbers of porcine pancreatic islets labeled with Resovist, a carboxydextran-coated SPIO, were transplanted into the kidney capsules of normal mice and imaged with a 3D FIESTA sequence using a 1.5T clinical MR scanner. Labeled (n = 3) and unlabeled (n = 2) islets were transplanted into the kidney capsules of streptozotocin-induced diabetic mice. Blood glucose levels and MR signal intensities were monitored for 30 days post-transplantation. RESULTS: There were no significant differences in viability or insulin secretion between labeled and unlabeled islets. A strong correlation (r2 > 0.94) was evident between the number of transplanted islets and T2 relaxation times quantified by MRI. Transplantation with labeled or unlabeled islets helped restore normal sustained glucose levels in diabetic mice, and nephrectomies induced the recurrence of diabetes. The MR signal intensity of labeled pancreatic islets decreased by 80% over 30 days. CONCLUSION: The transplantation of SPIO-labeled porcine islets into the kidney capsule of diabetic mice allows to restore normal glucose levels, and these islets can be visualized and quantified using a 1.5T clinical MR scanner.

In Vivo MR Imaging of Magnetically Labeled Mesenchymal Stem Cells in a Rat Model of Renal Ischemia

OBJECTIVE: This study was designed to evaluate in vivo MR imaging for the depiction of intraarterially injected superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells (MSCs) in an experimental rat model of renal ischemia. MATERIALS AND METHODS: Left renal ischemia was induced in 12 male Sprague-Dawley rats by use of the catheter lodging method. In vivo MR signal intensity variations depicted on T2*-weighted sequences were evaluated in both the left and right kidneys prior to injection (n = 2), two hours (n = 4), 15 hours (n = 2), 30 hours (n = 2) and 72 hours (n = 2) after injection of SPIO-labeled MSCs in both kidneys. Signal intensity variations were correlated with the number of Prussian blue stain-positive cells as visualized in histological specimens. RESULTS: In an in vivo study, it was determined that there was a significant difference in signal intensity variation for both the left and right cortex (40.8 +/- 4.12 and 26.4 +/- 7.92, respectively) and for both the left and right medulla (23.2 +/- 3.32 and 15.2 +/- 3.31, respectively) until two hours after injection (p < 0.05). In addition, signal intensity variation in the left renal cortex was well correlated with the number of Prussian blue stain-positive cells per high power field (r = 0.98, p < 0.05). CONCLUSION: Intraarterial injected SPIO-labeled MSCs in an experimental rat model of renal ischemia can be detected with the use of in vivo MR imaging immediately after injection.

Assessment of Lymph Node Metastases by Contrast-Enhanced MR Imaging in a Head and Neck Cancer Model

OBJECTIVE: We wanted to investigate the accuracy of contrast-enhanced MR imaging for the detection of lymph node metastases in a head and neck cancer rabbit model. MATERIALS AND METHODS: The metastatic lymph node model we used was created by inoculating VX2 tumors into the auricles of six New Zealand White rabbits. T1-weighted MR images were obtained before and after injecting gadopentetate dimeglumine at three weeks after tumor cell inoculation. The sizes, signal intensity ratios (i.e., the postcontrast signal intensities of the affected nodes relative to the adjacent muscle) and the enhancement patterns of 36 regional lymph nodes (parotid and caudal mandibular nodes) were evaluated on MR images and then compared with the histopathologic findings. RESULTS: No statistical difference was found between the sizes of 12 metastatic (10.5+/-3.2 mm) and 24 hyperplastic (8.0+/-3.6 mm) lymph nodes (p > 0.05). On the contrast-enhanced T1-weighted MR images, nine metastatic and four hyperplastic lymph nodes had peripheral high and central low signal intensity, whereas three metastatic and 20 hyperplastic lymph nodes had homogeneous high signal intensity. Using a signal intensity ratio less than one as a diagnostic criterion for a metastatic lymph node, the sensitivity, specificity and positive and negative predictive values of the enhanced MR images were 75% (9/12), 83% (20/24), 69% (9/13) and 87% (20/23), respectively, with areas under receiver-operating-characteristic curve values of 0.81. CONCLUSION: This experimental study confirms that metastatic and hyperplastic lymph nodes can be differentiated using MR images on the basis of the contrast uptake patterns, but that they cannot be differentiated using any particular size criteria.

Alteration of 4-aminopyridine-sensitive, voltage-dependent K+-channel in arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rats

Using the patch-clamp technique, we investigated the alteration of 4-aminopyridine(4-AP)-sensitive, voltage-dependent K+ channel (KV) in the mesenteric arterial smooth muscle cell (MASMC) of renovascular hypertensive model, one-kidney one-clip Goldblatt hypertensive rat (GBH). To isolate KV current, internal pipette solution contained 5 mM ATP and 10 mM EGTA. Under these condition, MASMC was depolarized by 4-AP, but charybdotoxin did not affect membrane potential. Membrane potential of hypertensive cell (- 40.3 +/- 3.2 mV) was reduced when compared to that of normotensive cell (-59.5 +/- 2.8 mV). Outward K+ current of hypertensive cell was significantly reduced when compared to normotensive cell. At 60 mV, the outward currents were 19.10 +/- 1.91 and 14.06 +/- 1.05 pA/pF in normotensive cell and hypertensive cell respectively. 4-AP-sensitive K+ current was also smaller in hypertensive cell (4.28 +/- 0.38 pA/pF) than in normotensive cell (7.65 +/- 0.52 pA/pF). The values of half activation voltage (V1/2) and slope factor (k1) as well as the values of half inactivation voltage (V1/2) and slope factor (k1) were virtually similar between GBH and NTR. These results suggest that the decrease of 4-AP-sensitive K+ current contributes to a depolarization of membrane potential, which leads to development of vascular tone in GBH.

Mechanism of membrane hyperpolarization by extracellular K+ in resistance-sized cerebral arterial muscle cell of rabbit

We sought to find out the mechanism of vascular relaxation by extracellular K+ concentration ((K+)o) in the cerebral resistant arteriole from rabbit. Single cells were isolated from the cerebral resistant arteriole, and using voltage-clamp technique barium-sensitive K+ currents were recorded, and their characteristics were observed. Afterwards, the changes in membrane potential and currents through the membrane caused by the change in (K+)o was observed. In the smooth muscle cells of cerebral resistant arteriole, ion currents that are blocked by barium, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) exist. Currents that were blocked by barium showed inward rectification. When the (K+)o were 6, 20, 60, and 140 mM, the reversal potentials were -82.7+/-1.0, -49.5+/-1.86, -26+/-1.14, -5.18+/-1.17 mV, respectively, and these values were almost identical to the calculated K+ equilibrium potential. The inhibition of barium-sensitive inward currents by barium depended on the membrane potential. At the membrane potentials of -140, -100, and -60 mV, Kd values were 0.44, 1.19, and 4.82 muM, respectively. When (K+)o was elevated from 6 mM to 15 mM, membrane potential hyperpolarized to -50 mV from -40 mV. Hyperpolarization by K+ was inhibited by barium but not by ouabain. When the membrane potential was held at resting membrane potential and the (K+)o was elevated from 6 mM to 15 mM, outward currents increased; when elevated to 25 mM, inward currents increased. Fixing the membrane potential at resting membrane potential and comparing the barium-sensitive outward currents at (K+)o of 6 and 15 mM showed that the barium-sensitive outward current increased at 15 mM K+. From the above results the following were concluded. Barium-sensitive K+ channel activity increased when (K+)o is elevated and this leads to an increase in K+ -outward current. Consequently, the membrane potential hyperpolarizes, leading to the relaxation of resistant arteries, and this is thought to contribute to an increase in the local blood flow of brain.

Decreased voltage dependent K+ currents in cerebral arterial smooth muscle cells of one-kidney, one-clip Goldblatt hypertensive rat

The Kv channel activity in vascular smooth muscle cell plays an important role in the regulation of membrane potential and blood vessel tone. It was postulated that increased blood vessel tone in hypertension was associated with alteration of Kv channel and membrane potential. Therefore, using whole cell mode of patch-clamp technique, the membrane potential and the 4-AP-sensitive Kv current in cerebral arterial smooth muscle cells were compared between normotensive rat and one-kidney, one-clip Goldblatt hypertensive rat (1K,1C-GBH rat). Cell capacitance of hypertensive rat was similar to that of normotensive rat. Cell capacitance of normotensive rat and 1K,1C-GBH rat were 20.8+/-2.3 and 19.5+/-1.4 pF, respectively. The resting membrane potentials measured in current clamp mode from normotensive rat and 1K,1C-GBH rat were -45.9+/-1.7 and -38.5+/-1.6 mV, respectively. 4-AP (5 mM) caused the resting membrane potential hypopolarize but charybdotoxin (0.1 muM) did not cause any change of membrane potential. Component of 4-AP-sensitive Kv current was smaller in 1K,1C-GBH rat than in normotensive rat. The voltage dependence of steady-state activation and inactivation of Kv channel determined by using double-pulse protocol showed no significant difference. These results suggest that 4-AP-sensitive Kv channels play a major role in the regulation of membrane potential in cerebral arterial smooth muscle cells and alterations of 4-AP-sensitive Kv channels would contribute to hypopolarization of membrane potential in 1K,1C-GBH rat.

Effect of pH on the ATP-sensitive K+ channel in aortic smooth muscle cells from rats

The effects of pH on K+ currents were investigated in single smooth muscle cells isolated from the thoracic aorta of Wistar-Kyoto rats. Whole-cell K+ currents were recorded in the conventional configuration of the voltage-clamp technique. Pinacidil (10muM) activated the whole-cell current and the pinacidil-activated current was completely inhibited by glibenclamide (10muM), an inhibitor of ATP-sensitive K+ channel (KATP channel). Pinacidil-activated current was reversed at near the K+ equilibrium potential. This current was time- and voltage-independent and reduced by elevating intracellular ATP. Pinacidil-activated current was reduced by lowering the external pH. However, alteration of internal pH has controversial effects on pinacidil-activated current. When the single cell was dialyzed with 0.1 mM ATP, alteration of internal pH had no effect on pinacidil-activated K+ current. In the contrast, when the single cell was dialyzed with 3 mM ATP, pinacidil-activated current was increased by lowering internal pH. Our results suggest that K+ channel activated by pinacidil may be KATP channel and internal H+ may reduce the inhibitory effect of ATP on KATP channel.