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Introduction Stress urinary incontinence (SUI) is the common type of urinary incontinence with a significantly negative impact on quality of life, including sexual dysfunction, in women. Although the etiology of SUI in women seems to be multifactorial, multiple vaginal parities and estrogen deficiency (ED) are considered to be major risk factors of SUI in elderly women. Thus, animal models of simulated birth trauma induced by multiple vaginal distention (VD) and ED induced by bilateral ovariectomy (OVX) have been used to study the SUI pathophysiology in rats. In previous studies, bilateral ovariectomy (OVX) reportedly induces the morphological changes in the rat urethral epithelium. It has also been reported that serotonergic (5HT+) paraneurons uniquely located in the urethra modulate urethral sensory function and that their numbers decrease in aged or diabetes rats. However, the difference of changes in the urethra such as those in epithelial morphology and expression of 5HT+ paraneurons in the urethra, in these two models has not been well characterized. Objective We therefore evaluated the histological changes of the urethral epithelium and 5HT+ cell expression, and urethral continence function in rats with OVX-induced ED or VD-induced simulated birth trauma. Methods Nulliparous SD rats were divided into: (1) sham group; (2) OVX 3-weeks group; (3) OVX 6-weeks group; (4) VD-1 group (1 time VD 2 weeks before assays); (5) VD-3 group (3 times VDs every 2 weeks). VD was induced by balloon catheter inflation for 4 hours in the vagina under isoflurane anesthesia. Urethral function was evaluated by the amplitude of urethral pressure responses during sneezing (A-URS) and urethral baseline pressure (UBP), which was measured by a microtransducer-tipped catheter inserted into the mid-urethra (n=6, each group). Following the urethral function analysis, the mid urethra was harvested, and the tissue sections (10 μm) were stained with Hematoxylin-eosin (H&E) and anti-5HT antibodies for immunofluorescent detection of 5HT+ paraneurons. In addition, in separate groups of rats, changes in urethral mRNA levels of tryptophan hydroxylase-1 (TPH1), a key enzyme of 5HT synthesis, were quantified by RT-PCR (n=6, each group). All data are represented as means ± SE. P < 0.05 was considered significant. Results OVX 6-weeks and VD-3 rats exhibited significant decreases in UBP and A-URS vs. sham (Fig. 1A). The mean epithelial thickness was significantly decreased in OVX 3, 6-weeks, VD-1 and 3 rats (Fig 1B,C) vs. sham. The number of urethral 5HT+ paraneurons was significantly decreased in OVX 3, 6 weeks, VD-1 and 3 rats (Fig. 2A,B) vs. sham. TPH-1 mRNA levels were significantly decreased in OVX 3, 6-weeks and VD-3 rats (Fig. 2C) vs. sham. Conclusions These results indicate that urethral epithelial atrophy and loss of 5HT+ paraneurons occur prior to SUI- inducing urethral dysfunction in both models, with the changes being rapid and greater in OVX compared to VD. These histological changes in the urethra may contribute to the establishment of SUI in ED and multiple birth trauma. Disclosure Work supported by industry: no.

Background: Survival outcomes are poor for patients with metastatic urothelial carcinoma who receive standard, first-line, platinum-based chemotherapy. We assessed the overall survival of patients who received durvalumab (a PD-L1 inhibitor), with or without tremelimumab (a CTLA-4 inhibitor), as a first-line treatment for metastatic urothelial carcinoma. Methods: DANUBE is an open-label, randomised, controlled, phase 3 trial in patients with untreated, unresectable, locally advanced or metastatic urothelial carcinoma, conducted at 224 academic research centres, hospitals, and oncology clinics in 23 countries. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0 or 1. We randomly assigned patients (1:1:1) to receive durvalumab monotherapy (1500 mg) administered intravenously every 4 weeks; durvalumab (1500 mg) plus tremelimumab (75 mg) administered intravenously every 4 weeks for up to four doses, followed by durvalumab maintenance (1500 mg) every 4 weeks; or standard-of-care chemotherapy (gemcitabine plus cisplatin or gemcitabine plus carboplatin, depending on cisplatin eligibility) administered intravenously for up to six cycles. Randomisation was done through an interactive voice–web response system, with stratification by cisplatin eligibility, PD-L1 status, and presence or absence of liver metastases, lung metastases, or both. The coprimary endpoints were overall survival compared between the durvalumab monotherapy versus chemotherapy groups in the population of patients with high PD-L1 expression (the high PD-L1 population) and between the durvalumab plus tremelimumab versus chemotherapy groups in the intention-to-treat population (all randomly assigned patients). The study has completed enrolment and the final analysis of overall survival is reported. The trial is registered with ClinicalTrials.gov, NCT02516241, and the EU Clinical Trials Register, EudraCT number 2015-001633-24. Findings: Between Nov 24, 2015, and March 21, 2017, we randomly assigned 1032 patients to receive durvalumab (n=346), durvalumab plus tremelimumab (n=342), or chemotherapy (n=344). At data cutoff (Jan 27, 2020), median follow-up for survival was 41·2 months (IQR 37·9–43·2) for all patients. In the high PD-L1 population, median overall survival was 14·4 months (95% CI 10·4–17·3) in the durvalumab monotherapy group (n=209) versus 12·1 months (10·4–15·0) in the chemotherapy group (n=207; hazard ratio 0·89, 95% CI 0·71–1·11; p=0·30). In the intention-to-treat population, median overall survival was 15·1 months (13·1–18·0) in the durvalumab plus tremelimumab group versus 12·1 months (10·9–14·0) in the chemotherapy group (0·85, 95% CI 0·72–1·02; p=0·075). In the safety population, grade 3 or 4 treatment-related adverse events occurred in 47 (14%) of 345 patients in the durvalumab group, 93 (27%) of 340 patients in the durvalumab plus tremelimumab group, and in 188 (60%) of 313 patients in the chemotherapy group. The most common grade 3 or 4 treatment-related adverse event was increased lipase in the durvalumab group (seven [2%] of 345 patients) and in the durvalumab plus tremelimumab group (16 [5%] of 340 patients), and neutropenia in the chemotherapy group (66 [21%] of 313 patients). Serious treatment-related adverse events occurred in 30 (9%) of 345 patients in the durvalumab group, 78 (23%) of 340 patients in the durvalumab plus tremelimumab group, and 50 (16%) of 313 patients in the chemotherapy group. Deaths due to study drug toxicity were reported in two (1%) patients in the durvalumab group (acute hepatic failure and hepatitis), two (1%) patients in the durvalumab plus tremelimumab group (septic shock and pneumonitis), and one (

Poster presentada en el 50th ERA-EDTA Congress (European Renal Association - European Dialysis Transplant Association), celebrado del 18 al 21 de mayo de 2013 en Estambul (Turquia)

As immunosuppressive therapy has advanced, we have markedly improved the outcome of ABO blood group incompatible living donor kidney transplantation. Consequently, graft survival at early phase after ABO-incompatible transplantation has been favorable than ABO-compatible transplantation in Japan. But in these days, it has been assumed that transplant glomerulopathy within one yr after ABO-incompatible kidney transplantation might be significantly precipitated. That may be because of chronic, active antibody-mediated rejection (AMR). We performed kidney graft biopsies at the early phase within 90 d after living donor kidney transplantation that involved the episode and protocol biopsies and studied findings of graft biopsy specimens when compared with ABO incompatible and compatible involving non-identical and identical transplantations. In ABO-incompatible transplant cases, the ratio occurring glomerulitis, especially severe injury of g 2-3, was significantly higher than that of identical and non-identical transplant cases (p < 0.01). There was no significant difference in t score, i score, ptc score and v score between three transplant groups. The cases occurring AMR were concordant with the cases recognized with severe glomerulitis. AMR was difficult to be diagnosed by C4d analysis in ABO-incompatible transplant cases. Glomerular injury score, g score, may be considered as more significant and the injury should be cured thoroughly.

Summary Background Tumour necrosis factor alpha is the key inflammatory cytokine involved in the pathogenesis of Crohn’s disease. Infliximab, a chimaeric monoclonal antibody of tumour necrosis factor-α is successfully used for the treatment of Crohn’s disease, although the response to infliximab therapy differs among patients. The genetic background of the individual may partially explain the differences of the responsiveness. Aim To investigate whether the polymorphisms in these genes are associated with the response to infliximab treatment as tumour necrosis factor-α exerts its biological activity through TNF receptor superfamily 1A and 1B. Methods Eighty Crohn’s disease patients were enrolled in the study and classified into responder and nonresponder according to the efficacy of infliximab treatment. Single nucleotide polymorphisms of TNF receptor superfamily 1A (rs767455 and rs4149570) and TNF receptor superfamily 1B (rs1061622, rs1061624 and rs3397) were determined. Results The minor allele carrier of rs767455 showed a significant association with a lack of efficacy compared to the major genotype (OR = 0.26; 95% CI: 0.08–0.91). A TNF receptor superfamily 1B haplotype inferred by rs1061624 and rs3397 also showed significant differences in the distribution between responder and nonresponder (P = 0.01). Conclusion These results suggest that tumour necrosis factor receptor genotypes may be involved in the different responses to infliximab in Japanese patients with Crohn’s disease.

Objective Hip pain and injury as a result of activity can lead to the development of early hip osteoarthritis (OA) in susceptible individuals. Our understanding of the factors that increase susceptibility continues to evolve. The ability to clearly identify individuals (and cohorts) with activity-related hip pain who are at risk of early hip OA is currently lacking. The purpose of this study was to gain expert consensus on which key clinical measures might help predict the risk of early hip OA in individuals presenting with activity-related hip pain. The agreed measures would constitute a standardised approach to initial clinical assessment to help identify these individuals. Methods This Dephi study used online surveys to gain concordance of expert opinion in a structured process of ‘rounds’. In this study, we asked ‘What outcome measures are useful in predicting hip OA in activity-related hip pain?’ The Delphi panel consisted of experts from sport and exercise medicine, orthopaedics, rheumatology, physiotherapy and OA research. Results The study identified key clinical measures in the history, examination and investigations (plain anteroposterior radiograph and femoroacetabular impingement views) that the panel agreed would be useful in predicting future risk of hip OA when assessing activity-related hip pain. The panel also agreed that certain investigations and tests (eg, MR angiography) did not currently have a role in routine assessment. There was a lack of consensus regarding the role of MRI, patient-reported outcome measures (PROMs) and certain biomechanical and functional assessments. Conclusions We provide a standardised approach to the clinical assessment of patients with activity-related hip pain. Assessment measures rejected by the Delphi panel were newer, more expensive investigations that currently lack evidence. Assessment measures that did not reach consensus include MRI and PROMs. Their role remains ambiguous and would benefit from further research.

高齢発症の重症筋無力症は少なく, 特に80歳以上の本邦報告例は, われわれの調べ得た範囲内では自験例を含めて5例のみであった.今回, 82歳の男性患者に拡大胸腺摘出術を施行し良好な結果を得たので報告する.鼻声と嚥下困難を主訴に近医受診し, 精査にて重症筋無力症 (Osserman IIB) と診断された.血液生化学的検査では抗アセチルコリンレセプター抗体 (以下抗AChR抗体) が2.9ng/mlと上昇している以外異常所見は認めなかった。胸部単純写真およびCTでは上行大動脈に接する前縦隔に辺縁平滑な径5×3cmの腫瘤影を認めた.胸腺腫を伴う重症筋無力症の診断のもと拡大胸腺摘出術を施行した.摘出腫瘍は径5×3×3cm, 淡黄色弾性軟で, 直径1.5cmの嚢胞を認めた.症状は術直後より著明に改善し, 術後4日目の抗AChR抗体は0.9ng/mlであった.術後経過は良好で合併症はなく症状はほぼ消失し, 第22病日に退院となった.

Hip fracture incidence is lower in Japan than in the West. Although differences have been found in peak bone mass and hip geometry between white and Japanese populations, these do not fully explain the difference in hip fracture rates. Variation in the rates of involutional bone loss may be an additional contributing factor. We address this issue in a prospective epidemiological study comparing bone loss rate among elderly people in Britain and Japan. Two population-based studies of bone loss rate in a British and a Japanese cohort were performed. Annual bone loss rates were obtained for 172 Hertfordshire men and 143 Hertfordshire women of mean age 66 years, and a questionnaire administered to obtain information on known confounding lifestyle factors. Eighty-six Japanese men and 90 Japanese women of mean age 69 years completed a similar study in Taiji, Japan. British men and women were heavier than Japanese men and women. Differences in lifestyle were also evident; the British men were less likely to smoke and the women more likely to consume alcohol than their Japanese counterparts. The British population also spent more time walking outdoors. Statistically significant differences between the two populations were apparent in baseline bone mineral density at lumbar spine (p < 0.05) and trochanter (p < 0.001) in men and women with Japanese subjects having lower values. There were also significant differences in bone density at the femoral neck (p < 0.001) between British and Japanese males. Men gained bone at the lumbar spine over the follow-up period in both populations. Bone loss rates were generally greater in the British female population than in Japanese women: the difference was statistically significant at the femoral neck (p < 0.05) and femoral trochanter (p < 0.001). These differences all remained significant after adjustment for differences in age between the two populations. Japanese subjects appear to have lower peak bone mass, but slower bone loss rates in later life than their European counterparts. These differences in bone loss rate help to explain the relatively low hip fracture rates found in Japan.

In the present study, it is observed that the tracking time of silicone rubber insulation under negative dc is less compared to positive dc voltage. It is noticed that the current flow during tracking studies is highly intermittent under the positive dc voltage compared to the negative dc voltage. It is realized that the energy content of the surface discharge current (wavelet transformed current signal) measured under the negative dc voltage is high compared to positive dc voltage. It is realized from wavelet transform study that the tracking resistance of the material and the discharge energy level of the current signal measured during tracking studies shows an inverse relationship. � 2006 IEEE.

Mecobalamin has been widely used in the treatment of patients with peripheral neuropathy. Several studies have investigated oral, intravenous (IV), or intramuscular administration of mecobalamin, but no studies on the usefulness of a combination of these routes have been reported. In addition, drug efficacy has been evaluated mainly from subjective symptoms, the severity of which is difficult to express numerically. In the present study, the effectiveness of two practical regimens of mecobalamin was investigated: (1) oral administration of 500 μg of mecobalamin three times a day for at least 8 weeks (O group); and (2) IV administration of 500 μg/d of mecobalamin three times a week for 4 weeks followed by oral administration of 500 μg of mecobalamin three times a day for at least 4 weeks (VO group). Not only were the changes in subjective symptoms and neurologic findings evaluated, but vibration perception thresholds were also monitored to evaluate the efficacy of each regimen as objectively as possible. Overall improvement was evaluated as "improved" or "markedly improved" in 66.6% of the VO group and 39.1% of the O group. Vibration perception thresholds of the lower extremities were significantly improved in patients in the VO group, whereas patients in the O group did not show clear improvement. The regimen was evaluated as "useful" or "highly useful" in 71.4% of the VO group and 43.5% of the O group. The investigators concluded that the VO regimen is highly effective, and it is recommended in patients who require more rapid and greater improvement of neurologic symptoms.

To evaluate promotive effects of hyperthermia on antitumor activity of new delta-alkyllactones (DALs) of low molecular weight (184-254 Da), chemically synthesized, which are different from natural macrocyclic lactones of high molecular weight (348-439 Da), such as camptothecin and sultriecin.A suspension of Ehrlich ascites tumor (EAT) cells was mixed with a DAL in a glass tube, heated at 37 or 42 degrees C for 30 min in a water bath, and cultured at 37 degrees C for 20 or 72 h. Cell viability was measured by the mitochondrial dehydrogenase- based WST-1 assay. DALs incorporated into EAT cells was extracted and measured by gas-liquid chromatography.The reduction of cell viability by DALs was markedly enhanced upon the treatment at 42 degrees C compared to that at 37 degrees C. At 37 degrees C, delta-hexadecalactone (DH16:0) and delta-tetradecalactone (DTe14:0) displayed cytostatic activity (at 100 microM survival level: 20.7%, 66.1%; at 50 microM--41.2%, 82.4%, respectively). Their activity was more marked at 42 degrees C (at 100 microM 10.6%, 27.6%; at 50 microM 30.6, 37.5%, ibid). The other DALs, delta-undecalactone (DU11:0), delta-dodecalactone (DD12:0), and delta-tridecanolactone (DTr13:0) were almost ineffective. Evaluation of survival rate in the cells treated for 30 min by DALs with the next culturing of EAT cells for 72 h resulted in the enhanced carcinostatic activity of DH16:0 and DTe14:0 even at concentrations as low as 25 microM at either 37 degrees C (18.5%, 78.5%, ibid) or 42 degrees C (5.0%, 42.0%, ibid), but the others exhibited slight activity or none. DH16:0 was effective at either 37 degrees C (36.0%) or 42 degrees C (23.0%) even at a lower dose of 10 microM. At the same time only the most cytostatic DH16:0 was incorporated into EAT cells and the rate of incorporation was more at 42 degrees C than at 37 degrees C.Delta-hexadecanolactone (DH16:0) exhibited the most cytostatic effect that was significantly enhanced by hyperthermia. It allows to consider it as a potent antitumor agent, especially in combination with hyperthermia.

Tumour necrosis factor alpha is the key inflammatory cytokine involved in the pathogenesis of Crohn's disease. Infliximab, a chimaeric monoclonal antibody of tumour necrosis factor-alpha is successfully used for the treatment of Crohn's disease, although the response to infliximab therapy differs among patients. The genetic background of the individual may partially explain the differences of the responsiveness.To investigate whether the polymorphisms in these genes are associated with the response to infliximab treatment as tumour necrosis factor-alpha exerts its biological activity through TNF receptor superfamily 1A and 1B.Eighty Crohn's disease patients were enrolled in the study and classified into responder and nonresponder according to the efficacy of infliximab treatment. Single nucleotide polymorphisms of TNF receptor superfamily 1A (rs767455 and rs4149570) and TNF receptor superfamily 1B (rs1061622, rs1061624 and rs3397) were determined.The minor allele carrier of rs767455 showed a significant association with a lack of efficacy compared to the major genotype (OR = 0.26; 95% CI: 0.08-0.91). A TNF receptor superfamily 1B haplotype inferred by rs1061624 and rs3397 also showed significant differences in the distribution between responder and nonresponder (P = 0.01).These results suggest that tumour necrosis factor receptor genotypes may be involved in the different responses to infliximab in Japanese patients with Crohn's disease.

The results of puncture breakdown tests on rubber gloves are presented. A positive polarity switching surge voltage (275/2500 mu s) or a 60 H alternating voltage was applied to the glove to cause breakdown. The effect of mechanical pressure on glove breakdown behavior and the influence of using a leather protector were investigated. It was found that protected gloves are more likely to break down at a position other than where the needle electrode has been placed. In both AC and switching surge breakdown tests, the standard deviation in breakdown voltage for protected samples was greater than that of the nonprotected samples by 25%. Under AC conditions, with mechanical stress applied, a bare glove will break down at a voltage which is approximately 22% below the nonstressed condition. A protected glove which is mechanically stressed will break down at a value 12.75% below the nonstressed condition. It is suggested that the results obtained will help to determine the operating limits and reliability of the glove insulation. >

Nakamura N., Yoshimura N., Ogi H., et al. Journal of Applied Physics, 118(8), 085302 (2015) https://doi.org/10.1063/1.4928959, Dynamics of continuous film formation of metallic films on quartz substrates is studied using an electrodeless resonance method. Bare quartz is used as a substrate, and a metallic film is deposited on it. We use antenna transmission technique to measure the evolution of resonance frequencies and internal friction of the substrate during and after deposition, and the morphological transition between discontinuous islands and a continuous film is detected. By comparison with atomic force microscopy images, we confirm that the frequency drop and the internal-friction peak that appear during deposition indicate this transition. We also find that Pt film shows unexpected morphology change after deposition.

In the present paper, the tracking phenomena of silicone rubber material has been studied under ac voltages, with ammonium chloride as a contaminant. The characteristic changes in the tracking time of the aged specimens are analyzed through leakage current measurement. The observed trends in the leakage current during the tracking study are analyzed using a moving average current technique. The contact angle of the specimen varies with the type of aging. multiresolution signal decomposition technique was adopted to identify the characteristic change in the hydrophobicity levels of the specimens using the standard deviation multiresolution analysis curve. It is concluded that the material with less contact angle where the leakage current magnitude is high, allows tracking to occur early. � 2006 IEEE.

Introduction A thorough literature review about the correlation of disc degeneration (DD) among the cervical, thoracic, and lumbar region revealed no studies that focused on a population-based cohort analysis of DD in the entire spine in humans using MRI. To further elucidate the role of intraindividual factors in the development of DD, we should examine DD in the entire spine. The purpose of this study, was to examine the correlation of DD in the cervical, thoracic, and lumbar region, respectively. Materials and Methods This was a cross-sectional study of a cohort from western Japan, comprising 975 (men, 324; women, 641) of 1,011 participants (age, 21-97 years). The participants were subjected to the entire spine MRI and all intervertebral discs in the cervical (C2/3-7/Th1), thoracic (Th1/2-Th12/L1), and lumbar (L1/2-L5/S) region on sagittal T2-weighted images were evaluated according to the Pfirrmann classification system (1-3= nondegeneration; 4, 5=degeneration). The Spearman rank correlation analysis was used to determine the correlation among the number of DD at each region mutually after adjustment for age. Results The Spearman rank correlation coefficient was 0.347 (95%CI: 0.5-0.59) between the cervical region and thoracic region, 0.300 (95%CI: 0.42-0.52) between the lumbar region and thoracic region, and 0.246 (95%CI: 0.34-0.44) between the cervical region and lumbar region. Conclusion Our population-based study using the whole spine MRI revealed a highly significant intraindividual correlation between DD in the cervical region and DD in the thoracic or lumbar region. Needless to say, the biomechanical environments among the three regions are fundamentally different. This distinct individual correlation for DD could be attributed to underlying genetic factor or endogenous factors, and so on. To further elucidate the role of intraindividual factors to DD, investigations on the extent of degenerative changes using the whole spine MRI could provide useful fundamental data concerning degenerative spinal change. Disclosure of Interest None declared

Introduction Intervertebral disc degeneration (DD) is thought to be the first step of degenerative spinal changes. Furthermore, DD is considered to be one of the causes of several symptoms including low back pain (LBP). Although previous studies have investigated the association between DD and LBP as well as risk factors of DD, many of them are still controversial mainly because of the selection bias for the cohort. The purposes of this study were (1) to examine the association between the presence of DD and low back pain, and (2) to examine the risk factors related to DD (age, gender, body mass index [BMI], hyperlipidemia, diabetes mellitus [DM], peripheral artery disease [PAD], and smoking) using a large scale population-based cohort. Materials and Methods This was a cross-sectional study of the base-line data from the population-based cohort of Wakayama, western part of Japan, comprising 975 (men, 324; women, 651; mean age 66.4 years) of 1,011 participants (age, 21-97 years). The participants were subjected to the entire spine MRI and the lumbar intervertebral disc was evaluated according to the Pfirrmann classification system (1-3 = nondegeneration; 4, 5 = degeneration). Multiple logistic regression analysis was used to estimate the association between DD and low back pain after adjustment for age, gender, and BMI. Besides this, we analyzed the association between the presence of DD and the potential risk factors: age, gender, BMI, hyperlipidemia, diabetes mellitus (DM), peripheral artery disease (PAD), and smoking. Results The presence of DD was significantly associated with low back pain. The multiple logistic regression analysis indicated that older age, obesity, and hyperlipidemia were significant related factors of DD. Gender, DM, PAD, and smoking were not significantly related with DD in our cohort. Conclusion The current data provide the foundation for elucidating the causes and mechanisms of DD. However, there is a limitation concerning a temporal relationship between exposure and outcome due to the nature of cross-sectional study design. The second survey of the cohort, which is now ongoing, will provide important information in the near future. Disclosure of Interest None declared Reference Teraguchi M, Yoshimura N, Hashizume H, et al. Prevalence and distribution of intervertebral disc degeneration over the entire spine in a population-based cohort: the Wakayama Spine Study. Osteoarthritis Cartilage 2014;22(1):104–110

We have observed weak expression of calponin h1, which stabilizes the actin filament system, in blood vessels within human malignant tumors. This observation suggested that because of a deficiency in stabilization by calponin h1, the structure of blood vessels in malignant tumors is fragile compared with blood vessels in normal tissues. We therefore generated calponin h1-deficient (CN(-/-)) mice to examine the effect of calponin h1 on the integrity of the barrier system in blood vessels against cancer metastasis. The CN(-/-) mice exhibited morphological fragility of the tissues, including the uterus and blood vessels. In particular, we frequently observed bleeding into the surrounding tissue from blood vessels of the ocular fundus in CN(-/-) mice. In addition, mesothelial cells, which usually express calponin h1 in normal (CN(+/+)) mice, were retracted in the CN(-/-) mice. When fluorescein was injected i.v. into mice, the CN(-/-) mice exhibited a greater and more rapid leakage of fluorescein from the blood vessels of the ocular fundus compared with the CN(+/+) mice. In the CN(-/-) mice receiving i.v. inoculations of B16 melanoma cells, significantly more metastatic nodules were formed in the lung than in the CN(+/+) mice. When B16 melanoma cells were injected i.p., the severity of peritonitis carcinomatosa was greater in CN(-/-) than in CN(+/+) mice. These results indicate that calponin h1 plays an important role in the regulation of the integrity of the blood vessels and peritoneum, which in turn is an important factor influencing the frequency of cancer metastasis. The CN(-/-) mice, which exhibit fragile blood vessels and peritoneum, could serve as sensitive and useful host models to investigate cancer metastasis.

To investigate whether lipid peroxides play a role in retinal cell death due to ischemia-reperfusion injury, whether recombinant human thioredoxin (rhTRX) treatment reduces production of lipid peroxides of the retina, and whether such treatment reduces the number of cells expressing c-Jun and cyclin D1.Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. After reperfusion, immunohistochemical staining for lipid peroxide, peroxynitrite, c-Jun, and cyclin D1 and propidium iodide (PI) staining were performed on retinal sections from animals treated intravenously with and without rhTRX, a free radical scavenger. Quantitative analyses of PI-, c-Jun-, and cyclin D1-positive cells were performed after the ischemic insult. Concentration of lipid peroxides in the retina was determined by the thiobarbituric acid assay.Specific immunostaining for lipid peroxides was seen in the ganglion cell layer at 6 hours after reperfusion, in the inner nuclear layer at 12 hours, and in the outer nuclear layer at 48 hours. Time course studies for PI-positive cells in the three nuclear layers coincided with those of specific immunostaining for lipid peroxides. The specific immunostaining was weakened by pre- and posttreatment with 0.5 mg of rhTRX. The number of PI-, c-Jun-, and cyclin D1-positive cells and the concentration of lipid peroxides were significantly decreased by treatment with rhTRX compared with those of vehicle-treated control rats (P:0. 01).Lipid peroxides formed by free radicals may play a role in neuronal cell death in retinal ischemia-reperfusion injury.

Retinal ischemia-reperfusion injury induces apoptosis of retinal neurons. The purpose of this study was to examine the association of c-Jun, caspase-1, -2, and -3 immunoreactivities and neuronal apoptosis in the retinal ganglion cell layer (GCL) and to study the effects of intravitreal brain-derived neurotrophic factor (BDNF) on the expression of these gene products in a rat model of retinal ischemia-reperfusion injury.After 60 minutes of ischemia, eyes were enucleated after 3, 6, 12, 24, and 168 hours of reperfusion. The numbers of c-Jun-, caspase-1-, caspase-2-, caspase-3, and TdT-dUTP terminal nick-end labeling (TUNEL)-positive cells in the GCL were counted. Recombinant human BDNF (5 microg) or vehicle was injected intravitreally immediately after reperfusion. At 6, 24, and 168 hours, the numbers of immunoreactive cells in BDNF- and vehicle-treated groups were compared.Expression of c-Jun and caspase-2 was found in dying cells in flat-mounted retinas. The numbers of caspase-1- and caspase-3-positive cells were fewer than c-Jun- or caspase-2-positive cells. Cell death in the retinal GCL was suppressed by an intravitreal injection of BDNF. The numbers of TUNEL- and caspase-2-positive cells were lower in the BDNF-treated group at 6 hours after reperfusion (P0.01). The number of c-Jun-positive cells in the treated retinas was not altered by the treatment.Expression of c-Jun and caspase-2 is associated with neuronal cell apoptosis in the GCL. Suppression of caspase-2 expression may explain the neuroprotective effects of BDNF.

To evaluate possible roles of caspase-1 and caspase-3 in retinal ischemia-reperfusion injury.Retinal ischemia was induced in rats by increasing the intraocular pressure to 110 mm Hg for 60 minutes. Expression of caspase-1 and caspase-3 was studied at the mRNA and protein levels using immunohistochemical staining, western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and assay of the enzymatic activities. Apoptotic retinal neurons were detected by the TdT-dUTP terminal nick-end labeling (TUNEL) method. To study the roles of the caspases in retinal ischemia-reperfusion injury, an inhibitor of caspase-1, acetyl-tyrosyl-valyl-alanyl-aspart-1-al (Ac-YVAD-CHO; total dose, 10(-7) moles) and that of caspase-3, acetyl-aspartyl-glutamylvalyl-aspart-1-al (Ac-DEVD-CHO; total dose, 10(-7) moles) was injected intravitreally and the number of TUNEL-positive cells was compared with the number in sections not treated with the inhibitors.In the inner nuclear layer (INL), caspase-3-like immunoreactivity was predominantly detected, whereas caspase-1-like immunoreactivity was more predominant in the outer nuclear layer (ONL). Expression of caspase-1 and -3 was upregulated at the protein and gene levels 24 hours after reperfusion. Intravitreal injection of Ac-DEVD-CHO decreased the number of TUNEL-positive cells more significantly in the INL than in the ONL (P0.01) at 24 hours, whereas, intravitreal injection of Ac-YVAD-CHO was more effective in decreasing the number in the ONL (P0.05).These findings suggest a possibility that cell-type-specific activation of caspases takes place in retinal ischemia-reperfusion injury, and such caspase may induce retinal neuronal cell death.

To study the role of caspase-like proteases, especially roles of more extensively characterized caspase-1 and caspase-2, in apoptotic photoreceptor cell degeneration in Royal College of Surgeons (RCS) rats.Both RCS and Sprague-Dawley rats were used. Cryosections of the retinas at various postnatal times were immunostained with antibodies against caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-2 (Nedd2/Ich-1). Double staining with TdT-dUTP terminal nick-end labeling (TUNEL), propidium iodide, and the antibodies was also performed. To evaluate the time course of protein expression, western blot analysis was carried out. The temporal profile of caspase-like protease activity was studied using a fluorogenic tetrapeptide substrate, acetyl-tyrosyl-valyl-alanyl-aspartic acid alpha (4-methyl-coumaryl-7-amide) (Ac-YVAD-MCA). Intravitreal injection of a caspase-1 inhibitor, acetyl-tyrosyl-valyl-alanyl-aspartic-aldehyde (Ac-YVAD-CHO), at postnatal days 21 (P21) and P26 was performed to see if this caused a decrease in apoptotic cell number at P28.TUNEL-positive photoreceptors of RCS rats stained strongly with antibodies against caspase-1 and caspase-2. Double staining studies revealed that caspase-1 and caspase-2 were coexpressed in apoptotic cells. Western blot analysis showed that active forms of caspase-1-like and caspase-2-like proteases were upregulated at P28, concurrent with the peak in TUNEL-positive cells. The enzymatic activity of caspase-1-like protease was elevated in RCS rat retinas at P28, and the inhibitor of caspase-1 transiently reduced the number of the apoptotic photoreceptors.Activation of caspase-like proteases plays an important role in photoreceptor apoptosis of RCS rats.

To determine whether an angiogenic inhibitor, TNP- 470 (TNP), an analogue of fumagillin, inhibits choroidal neovascularization (CNV) induced by diode laser photocoagulation in a rat experimental model.Fundus laser photocoagulation was performed on Brown Norway rats to induce CNV. In the treatment group, TNP was administered intraperitoneally at the time of laser photocoagulation and on day 7 (50 mg/kg at each time). The incidence of CNV formation was evaluated by fluorescein angiography. The retina was collected from the rats on days 1, 3, 7, and 14 after laser photocoagulation, and semiquantitative polymerase chain reaction (PCR) analyses for the expression of mRNA of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were carried out. Localization of bFGF mRNA was studied by in situ reverse transcription-PCR (RT-PCR). The numbers of positively labeled cells for bFGF mRNA were compared between the TNP treatment and control groups.The incidence of CNV formation was 22.7% in the TNP-treated rats and that in the control rats was 61.4% (P0.001). The semiquantitative PCR analyses showed that bFGF mRNA was upregulated on days 3 and 7 in the control rats, but no significant changes were found in TNP-treated rats. There was no detectable difference in VEGF gene expression between the control and TNP-treated rats. bFGF mRNA was detected by in situ RT-PCR in the regenerated retinal pigment epithelial cells and cells of the outer and inner nuclear layers of the control rats. The number of positive cells for bFGF mRNA in the TNP treatment group was significantly smaller than that of the control group (P0.05) on days 3 and 14.TNP- 470 treatment reduced the incidence of laser-induced CNV formation in this experimental model. The expression of bFGF associated with CNV formation was also significantly reduced by the TNP treatment.

The effects of ligustrazine on hepatic oxygenation in the isolated rat liver were investigated during prolonged perfusion and following the injection of norepinephrine. After injection of erythrocytes into the perfusate, the hemoglobin spectra in the liver were measured by Erlangen microlightguide spectroscopy, and the hemoglobin oxygenation (HbO2) in the liver was calculated on the basis of the Kubelka-Munk theory. During artificial perfusion, the HbO2 value was decreased from 59.3 +/- 6.4% (after one hour's perfusion) to 25.5 +/- 19.5% (n = 441; after six hours' perfusion). However, when ligustrazine was injected into the perfusate after six hours' perfusion, the HbO2 values recovered to 56.4 +/- 9.7% (n = 441). After injection of norepinephrine, HbO2 in the liver decreased from 48.8 +/- 10.4% to 25.2 +/- 18.4% (n = 961), while subsequent administration of ligustrazine caused a recovery to 62.9 +/- 6.0% (n = 961). Our results suggested that ligustrazine is a powerful hepatic vasodilator for improving hepatic oxygenation.

The aim of this study was to identify a method to determine whether segmental artery reconstruction was indicated during aortic clamping.Spinal cord evoked potential (SCEP) and regional spinal blood flow were studied in 24 adult dogs. Using the left heart bypass technique, aortic clamping divided the aorta into thoracic, abdominal, and terminal segments. After the occlusion of the descending aorta and discontinuation of the perfusion to the abdominal segment, animals were assigned to four groups: no treatment with perfusion to the terminal aorta (group IA), no treatment with cessation of bypass (group IB), 5% lidocaine administration (5 mg/kg) into the abdominal segment with perfusion to the terminal aorta (group IIA), and lidocaine administration with cessation of bypass (group IIB).Cessation of bypass reduced spinal blood flow and SCEP amplitude. Lidocaine injection allowed for a significant rapid decrease in SCEP amplitude in group IIB compared with group IB (24.2% +/- 13.4% versus 92.3% +/- 33.2%; p = 0.0039). The degree of spinal blood supply was reflected immediately in the magnitude of SCEP amplitude change by the direct lidocaine injection. (group IB versus group IIB; p = 0.023).The direct injection of lidocaine into the clamped aorta results in a rapidly change in SCEP in the threat of ischemia and can be used to make an early detection of the segmental arteries perfusing to the spinal cord.

To determine whether tranilast administered to pigmented rats inhibits formation of choroidal neovascularization induced by diode-laser photocoagulation.Female Brown Norway rats were used. On day 0, choroidal neovascularization was induced by diode-laser photocoagulation, using a setting of 75 microm spot size, 0.1 second's duration, and 100 mW intensity. Tranilast (200 or 600 mg/kg per day) was administered orally twice daily for 14 days. Indomethacin (1 and 5 mg/kg per day) was administered orally once a day for 14 days. Choroidal neovascularization was evaluated on days 7 and 14 by fundus photography and fluorescein angiography. Late-phase fluorescein angiography was scored according to four grades. The animals were killed on day 14, and the lesions were evaluated histologically.In the vehicle-treated group, 34 of 35 burns (97%) showed fluorescein staining and late leakage on day 14. Choroidal neovascularization was identified by light microscopy in all the lesions that showed fluorescein staining and late leakage. The score of fluorescein staining was reduced in rats given 200 mg/kg per day or 600 mg/kg per day (P0.01) of tranilast. The thickness of the laser-induced lesions was reduced in a dose-dependent manner by tranilast, a significant difference was observed with 600 mg/kg per day (P0.05). Oral indomethacin treatment did not reduce fluorescein staining on day 14.Tranilast inhibits the development of choroidal neovascularization in this experimental model.

To study whether cell type-specific death occurs in retinal ischemia-reperfusion injury and the possible roles of p16INK4 in the determination of cell death.Retinal ischemia-reperfusion injury was induced in rats by a ligation method. After 1 hour of ischemia and a time of reperfusion that varied, rat eyes were enucleated. Cell death in the retina was studied by the TdT-dUTP terminal nick-end labeling method and propidium iodide (PI) staining. Electron microscopic observation of the retina was also performed. Immunohistochemical studies using antibodies against syntaxin and calbindin were performed to detect amacrine cells and horizontal cells, respectively, and immunohistochemical studies using an antibody against p16INK4 were performed to study whether this cell cycle-related protein was expressed in dying cells.Most of the calbindin-positive horizontal cells in the outer aspect of the inner nuclear layer (INL) showed morphologic features of necrosis. In contrast, syntaxin-positive amacrine cells in the inner aspect of the INL showed features of apoptosis. Of 320 calbindin-positive horizontal cells, only 11 (3.4%) showed positive PI staining. Those calbindin-positive, horizontal cells were p16INK4 positive. In contrast, 746 of 910 (82.0%) syntaxin-positive amacrine cells showed condensed PI staining, and none were p16INK4 positive.Expression of p16INK4 may regulate the fate of retinal neurons in ischemia-reperfusion injury, and cell type-specific death thus occurs in the retina after such injury.

Nitric oxide is a reactive species that could be protective or destructive to the retina depending on the stage of the evolving ischemic process. This study was conducted to obtain a better understanding of the roles of constitutive nitric oxide synthase (cNOS) during reperfusion after ischemia in rat retina.Retinal ischemia was induced for 60 minutes in Sprague-Dawley rats by ligating the optic nerve. Gene expression for endothelial and neuronal nitric oxide synthases (eNOS and nNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). To inhibit cNOS, NG-nitro-L-arginine (L-NNA) was injected intraperitoneally four times (every 6 hours) beginning 2 hours after reperfusion, for a total dose of 80 mg/kg. Retinal damage was assessed by the rate of a- and b-wave recovery on electroretinograms and by the thickness of the retinal layers. Retinal circulation and vessel diameter were evaluated by the dye-dilution technique.After ischemia ended, eNOS mRNA initially decreased until 6 hours, then increased to a peak at 12 hours, and decreased progressively beyond 24 hours until the final measurement at 96 hours of reperfusion. nNOS mRNA decreased to nearly undetectable levels during the same measurement periods. L-NNA treatment enhanced reduction of a- and b-wave amplitudes and increased thinning of the inner retina in postischemic eyes. Retinal mean circulation time was markedly prolonged in L-NNA-treated postischemic eyes. Arterial mean transit times were 2.1-fold and 4.5-fold longer in L-NNA-treated postischemic eyes than in L-NNA-treated nonischemic eyes and in D-NNA-treated postischemic eyes, respectively.This study shows that postischemic inhibition of NOS worsens retinal damage after ischemia-reperfusion and alters postischemic retinal circulation. Nitric oxide may play an important role in protecting the retina from ischemic injury, possibly by preventing postischemic hypoperfusion.