Development and Evaluation of a Short-time Imaging Method for the Clinical Study of the Apparent Diffusion Coefficient Subtraction Method.

The apparent diffusion coefficient subtraction method (ASM) was developed as a new restricted diffusionweighted imaging technique for magnetic resonance imaging (MRI). The usefulness of the ASM has been established by in vitro basic research using a bio-phantom, and clinical research on the application of the ASM for the human body is needed. Herein, we developed a short-time sequence for ASM imaging of the heads of healthy volunteers (n=2), and we investigated the similarity between the obtained ASM images and diffusion kurtosis (DK) images to determine the utility of the ASM for clinical uses. This study appears to be the first to report ASM images of the human head. We observed that the short-time sequence for the ASM imaging of the head can be scanned in approx. 3 min at 1.5T MRI. The noise reduction effect of median filter processing was confirmed on the ASM images scanned by this sequence. The obtained ASM images showed a weak correlation with the DK images, indicating that the ASM images are restricted diffusion-weighted images. The new shorttime imaging sequence could thus be used in clinical studies applying the ASM.

Evaluation of Fast Diffusion Kurtosis Imaging Using New Software Designed for Widespread Clinical Use.

Clinical research using restricted diffusion-weighted imaging, especially diffusion kurtosis (DK) imaging, has been progressing, with reports on its effectiveness in the diagnostic imaging of cerebral infarctions, neurodegenerative diseases, and tumors, among others. However, the application of DK imaging in daily clinical practice has not spread because of the long imaging time required and the use of specific software for image creation. Herein, with the aim of promoting clinical research using DK imaging at any medical facility, we evaluated fast DK imaging using a new software program. We developed a new macro program that produces DK images using general-purpose, inexpensive software (Microsoft Excel and ImageJ), and we evaluated fast DK imaging using bio-phantoms and a healthy volunteer in clinical trials. The DK images created by the new software with diffusion-weighted images captured with short-time imaging sequences were similar to the original DK images captured with long-time imaging sequences. The DK images using three b-values, which can reduce the imaging time by 43%, were equivalent to the DK images using five b-values. The DK imaging technique developed herein might allow any medical facility to increase its daily clinical use of DK imaging and easily conduct clinical research.

Investigation into the Effect of Breast Volume on Irradiation Dose Distribution in Asian Women with Breast Cancer.

Reports on irradiation dose distribution in breast cancer radiotherapy with sufficient sample size are limited in Asian patients. Elucidating dose distribution in Asian patients is particularly important as their breast volume differs compared to patients in Europe and North America. Here, we examined dose distribution in the irradiation field relative to breast volume for three irradiation methods historically used in our facility. We investigated the influence of breast volume on each irradiation method for Asian women. A total of 573 women with early-stage breast cancer were treated with breast-conserving surgery and adjuvant radiotherapy. Three methods were compared: wedge (W), field-in-field (FIF), and wedge-field-in-field (W-FIF). In patients with small breast volume, FIF decreased low- and high-dose areas within the planning target volume, and increased optimal dose area more than W. In patients with medium and large breast volumes, FIF decreased high-dose area more than W. The absolute values of correlation coefficients of breast volume to low-, optimal-, and high-dose areas and mean dose were significantly lower in FIF than in W. The correlation coefficients of V107% were 0.00 and 0.28 for FIF and W, respectively. FIF is an excellent irradiation method that is less affected by breast volume than W in Asian breast cancer patients.

Evaluation of the Imaging Process for a Novel Subtraction Method Using Apparent Diffusion Coefficient Values.

Diffusion-weighted imaging may be used to obtain the apparent diffusion coefficient (ADC), which aids the diagnosis of cerebral infarction and tumors. An ADC reflects elements of free diffusion. Diffusion kurtosis imaging (DKI) has attracted attention as a restricted diffusion imaging technique. The ADC subtraction method (ASM) was developed to visualize restricted diffusion with high resolution by using two ADC maps taken with different diffusion times. We conducted the present study to provide a bridge between the reported basic ASM research and clinical research. We developed new imaging software for clinical use and evaluated its performance herein. This software performs the imaging process automatically and continuously at the pixel level, using ImageJ software. The new software uses a macro or a plugin which is compatible with various operating systems via a Java Virtual Machine. We tested the new imaging software's performance by using a Jurkat cell bio-phantom, and the statistical evaluation of the performance clarified that the ASM values of 99.98% of the pixels in the bio-phantom and physiological saline were calculated accurately (p<0.001). The new software may serve as a useful tool for future clinical applications and restricted diffusion imaging research.

Evaluation of calculation processes of apparent diffusion coefficient subtraction method (ASM) imaging.

A number of restricted diffusion (RD) imaging techniques, such as diffusion kurtosis (DK) imaging and Q space imaging, have been developed and proven to be useful for the diagnosis of diseases, including cerebral gliomas and cerebrovascular infarction. In particular, apparent diffusion coefficient (ADC) subtraction method (ASM) imaging has become available recently as a novel RD imaging technique. ASM is based on the difference between the ADC values in an image pair of two ADC maps, ADC basic (ADCb) and ADC modify (ADCm), which are created from diffusion-weighted images taken using short and long effective diffusion times, respectively. The present study aimed to assess the potential of different types of ASM imaging by comparing them with DK imaging which is the gold-standard RD imaging technique. In the present basic study using both polyethylene glycol phantom and cell-containing bio-phantom, three different types of ASM images were created using different calculation processes. ASM/A is an image calculated by dividing the absolute difference between ADCb and ADCm by ADCb several times. By contrast, ASM/S is an image created by dividing the absolute difference between ADCb and ADCm by the standard deviation of ADCb several times. As for positive ASM/A image (PASM/A), the positive image, which was resultant after subtracting ADCb from ADCm, was divided by ADCb several times. A comparison was made between the types of ASM and DK images. The results showed the same tendency between ASM/A in addition to both ASM/S and PASM/A. By increasing the number of divisions by ADCb from three to five times, ASM/A images transformed from DK-mimicking to more RD-sensitive images compared with DK images. These observations suggest that ASM/A images may prove useful for future clinical applications in RD imaging protocols for the diagnosis of diseases.

Loss of P2Y1 receptors triggers glaucoma-like pathology in mice.

BACKGROUND AND PURPOSE: Glaucoma, the leading cause of blindness, damages the retinal ganglion cells. Elevated intraocular pressure (IOP) is a high-risk factor for glaucoma, so topical hypotensive drugs are usually used for treatment. Because not all patients do not respond adequately to current treatments, there is a need to identify a new molecular target to reduce IOP. Here, we have assessed the role of P2Y1 receptors in mediating elevated IOP. EXPERIMENTAL APPROACH: P2Y1 receptor agonist was instilled into the eyes of mice, and the IOP changes were measured by a rebound-type tonometer. Expression of P2Y1 receptors was estimated by immunohistochemistry. Ocular function was measured by a multifocal electroretinogram. KEY RESULTS: A single dose of the P2Y1 receptor agonist transiently reduced IOP and such effects were absent in P2Y1 receptor-deficient (P2Y1 KO) mice. P2Y1 receptors were functionally expressed in the ciliary body, trabecular meshwork and Schlemm's canal. Activation of P2Y1 receptors negatively regulated aquaporin 4 (AQP4) function but up-regulated endothelial NOS (eNOS). P2Y1 KO mice showed chronic ocular hypertension regardless of age. P2Y1 KO mice at 3 months old showed no damage to retinal ganglion cells, whereas 12-month-old mice showed a significant loss of these cells and impairment of ocular functions. Damage to retinal ganglion cells was attenuated by chronic administration of an IOP-reducing agent. CONCLUSION AND IMPLICATIONS: Activation of P2Y1 receptors reduced IOP via dual pathways including AQP4 and eNOS. Loss of P2Y1 receptors resulted in glaucomatous optic neuropathy, suggesting that P2Y1 receptors might provide an effective target in the treatment of glaucoma.

New field-in-field with two reference points method for whole breast radiotherapy: Dosimetric analysis and radiation-induced skin toxicities assessment.

The usefulness of the field-in-field with two reference points (FIF w/ 2RP) method, in which the dose reference points are set simultaneously at two positions in the irradiation field and the high-dose range is completely eliminated, was examined in the present study with the aim of decreasing acute skin toxicity in adjuvant breast radiotherapy (RT). A total of 573 patients with breast cancer who underwent postoperative whole breast RT were classified into 178 cases with wedge (W) method, 142 cases with field-in-field without 2 reference points (FIF w/o 2RP) method and 253 cases with FIF w/ 2RP method. Using the FIF w/ 2RP method, the high-dose range was the lowest among the three irradiation methods. The planning target volume (PTV) V105% and the breast PTV for evaluation (BPe) V105% decreased to 0.09 and 0.10%, respectively. The FIF w/ 2RP method vs. the FIF w/o 2RP method had a strong association (η) with PTV V105% (η=0.79; P<0.001) and BPe V105% (η=0.76; P<0.001). The FIF w/ 2RP method had a significant impact on lowering the skin toxicity grade in weeks 3 and 4, and increasing the occurrence of skin toxicity grade 0. The FIF w/ 2RP method vs. the W method had a moderate association with skin toxicity grade at week 3 (η=0.49; P<0.001). Using the FIF w/ 2RP method, the high-dose range V105% of the target decreased to 0%, and skin adverse events were decreased in conjunction. For patients with early-stage breast cancer, particularly patients with relatively small-sized breasts, the FIF w/ 2RP method may be an optimal irradiation method.

Development of a novel phantom using polyethylene glycol for the visualization of restricted diffusion in diffusion kurtosis imaging and apparent diffusion coefficient subtraction method.

The present study aimed to investigate whether polyethylene glycol (PEG) phantoms have the potential to be used as standard phantoms for magnetic resonance imaging (MRI) in order to visualize restricted diffusion in diffusion kurtosis imaging (DKI), the ADC subtraction method (ASM) and the apparent diffusion coefficient (ADC). Diffusion-weighted images of 0-120 mM PEG phantoms were captured to create ADC, DKI and ASM images with post-processing. ASM is a recently developed method for restricted diffusion imaging using the readout segmentation of long variable echo-train sequences. As the PEG concentration increases, the ADC value decreases. Conversely, an increase in DKI and ASM values is associated with increasing PEG concentration. Formulae were constructed to represent the association between PEG concentrations and ADC, DKI and ASM values. These formulae can be used to determine the required PEG concentrations to mimic arbitrary ADC, DKI and ASM values of certain diseases, including tumors and infarctions. Validation experiments were conducted using bio-phantoms and clarified that the PEG phantoms cover the range of ADC and DKI values reported in previous clinical research using 3T MRI. PEG phantoms may be useful for future MRI research involving restricted diffusion.

Risperidone attenuates local and systemic inflammatory responses to ameliorate diet-induced severe necrotic pancreatitis in mice: it may provide a new therapy for acute pancreatitis.

In a previous article, we showed that a potent serotonin-, 5-hydroxytryptamine-2A (5-HT(2A)) antagonist, risperidone, ameliorated cerulein-induced edematous pancreatitis in mice. In the present article, young female mice were fed a choline-deficient, ethionine-supplemented diet. All of the mice developed severe necrotic pancreatitis, and approximately 50% of them died within 4 days. Serum levels of proinflammatory interleukin (IL)-6 significantly increased on day 3 and returned toward the control on day 4 of choline-deficient ethionine-supplemented (CDE) diet treatment. The time course of IL-6 levels paralleled those of plasma amylase and lipase activities. On the other hand, platelet counts significantly decreased on day 3, and the change became more marked on day 4, coinciding with mortality and histological alterations of the pancreas (edema, inflammatory cell infiltration, necrosis). Preceding these changes, plasma levels of 5-hydroxyindoleacetic acid (5-HIAA) increased on feeding a CDE diet to reach a peak on day 3 and returned toward the control on day 4. Risperidone (0.1-3.2 mg/kg twice a day) hardly affected the 5-HIAA levels but dose-dependently attenuated the serum IL-6 levels, plasma amylase/lipase levels, platelet counts, histological alterations, and mortality of diet-induced pancreatitis mice. These results are discussed in relation to the pathogenesis of acute pancreatitis. Thus, we speculate that acinar cell injury triggers local inflammatory reactions and, if coincided with enhanced IL-6 release, leads to a systemic inflammatory response syndrome, which is responsible for the mortality. In addition, it is suggested that diet-induced 5-HT release and 5-HT(2A) receptor activation are involved in this whole process of pancreatitis development. Risperidone may provide a new therapy for the disease.

Development of a novel method for visualizing restricted diffusion using subtraction of apparent diffusion coefficient values.

In order to visualize restricted diffusion, the present study developed a novel method called 'apparent diffusion coefficient (ADC) subtraction method (ASM)' and compared it with diffusion kurtosis imaging (DKI). The diffusion-weighted images of physiological saline, in addtion to bio-phatoms of low cell density and the highest cell density were obtained using two sequences with different effective diffusion times. Then, the calculated ADC values were subtracted. The mean values and standard deviations (SD) of the ADC values of physiological saline, low cell density and the highest cell density phantoms were 2.95±0.08x10­3, 1.90±0.35x10­3 and 0.79±0.05x10­3 mm2/sec, respectively. The mean kurtosis values and SD of DKI were 0.04±0.01, 0.44±0.13 and 1.27±0.03, respectively. The ASM and SD values were 0.25±0.20x104, 0.51±0.41x104 and 4.80±4.51x104 (sec/mm2)2, respectively. Using bio­phantoms, the present study demonstrated that DKI exhibits restricted diffusion in the extracellular space. Similarly, ASM may reflect the extent of restricted diffusion in the extracellular space.

In vitro screening for antihyperlipidemic activities in foodstuffs by evaluating lipoprotein profiles secreted from human hepatoma cells.

We screened the antihyperlipidemic effects of seven edible plants by evaluation of the triglyceride (TG) and cholesterol profiles secreted from HepG2 cells. We found that the water- and ethanol-extracts of Brasenia schreberi at 100 µg/ml exhibited strong inhibitory activities against TG and cholesterol secretions from HepG2 cells stimulated with sodium oleate. Real-time RT-PCR analysis demonstrated that ethanol extract of B. schreberi (BSET) attenuated the expression of the sterol regulatory element binding protein-1c and -2, fatty acid synthase and HMG CoA synthase-1 genes, which are involved in lipid synthesis in hepatocyte/hepatoma cells. Furthermore, we studied the action of BSET on adipose tissue accumulation and serum parameters in mice fed a high-fat diet (HFD). BSET suppressed mesenteric and epididymal adipose tissue accumulation and normalized serum TG and glucose, but not cholesterol levels in HFD-fed mice.

Unidirectional binding of clostridial collagenase to triple helical substrates.

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca(2+). We have shown that CBD can be functional to anchor growth factors in local tissue. A (1)H-(15)N HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-l-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-ray scattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.

Possible involvement of endogenous 5-HT in aggravation of cerulein-induced acute pancreatitis in mice.

The aim of the present study was to elucidate the pathogenic role of endogenous 5-HT in pancreatitis. Injections of cerulein at hourly intervals caused edematous pancreatitis in mice characterized by hyperenzymemia and histological alterations. While the cerulein-induced hyperenzymemia was attenuated in mice pretreated with p-CPA, a 5-HT depletor, it was exaggerated by the preferential 5-HT2A agonist (DOI), but not by the preferential 5-HT2B agonist (BW723C86) or the preferential 5-HT2C agonist (mCPP). Selective 5-HT2A antagonists (risperidone, spiperone, ketanserin, AMI-193, and MDL 11,939) dose-dependently attenuated the hyperenzymemia; and their potency order, excepting that of ketanserin which has considerable affinity at the 5-HT2C receptor as well, paralleled their reported pKi values at the 5-HT2A receptor. Selective 5-HT2B (SB204741) and 5-HT2C (SB242084) antagonists hardly affected the hyperenzymemia. Although the non-selective 5-HT2A/2B/2C antagonists (metergoline, ritanserin, and methysergide) dose-dependently attenuated the hyperenzymemia, they were relatively less potent compared to their high pKi values at the 5-HT2A receptor. In another set of experiments, risperidone, but not SB204741 and SB242084, dose-dependently reversed the cerulein-induced histological alteration of the pancreas (inflammatory cell infiltration). These results suggest that endogenously released 5-HT activates 5-HT2A receptors to aggravate cerulein-induced pancreatitis. We propose that selective 5-HT2A antagonists may provide a new therapy for acute pancreatitis.