Self-harm and Suicide Attempts in a Japanese Psychiatric Hospital.

OBJECTIVE: Self-harm and attempted suicide are risk factors for suicide in psychiatric hospital in-patients. This study aimed to analyse the circumstances of self-harm and suicide attempts in a Japanese psychiatric hospital so as to improve management and care. METHODS: Incident reports of self-harm and suicide attempts during a 12.4-year period from November 2000 to March 2013 were reviewed. A descriptive analysis was conducted in terms of age, sex, and diagnosis of patients, as well as level, ward, situations, and causes of incidents. RESULTS: During the study period, 90 cases of self-harm and attempted suicide involving 58 patients were reported. The rate of self-harm and suicide attempts was 0.05 per 1000 patient-days. The types of selfharm and suicide attempts included hanging (n = 25), wrist cutting (n = 19), ingestion of foreign objects (n = 17), and others (n = 29). The single case of completed suicide involved hanging, in a patient with schizophrenia. Among 55 patients with relevant data, the most common clinical diagnosis was mood disorder (41.8%), followed by schizophrenia (36.4%). Mood disorder was 3.5 times as prevalent in females as in males (14 vs. 4). Fourteen patients with mood disorder (n = 8) or schizophrenia (n = 6) were repeatedly involved in 46 of 89 cases of self-harm or attempted suicide; 11 were female. One woman with mood disorder attempted suicide 9 times within the same year. The top 3 management and care factors related to self-harm and suicide attempts were failure to adhere to preventive procedures (28%), insufficient therapeutic communication (28%), and difficulty in predicting suicide (20%). CONCLUSION: Self-harm and suicide attempts at this psychiatric hospital occurred at a rate of 0.05 per 1000 patient-days between late 2000 and early 2013. Efforts are needed to increase compliance with suicide prevention procedures and therapeutic communication, so as to improve management and care of psychiatric in-patients and prevent them from committing suicide.

Positive association of free triiodothyronine with pancreatic ß-cell function in people with prediabetes.

AIM: To analyse the effects of thyroid hormones on ß-cell function and glucose metabolism in people with prediabetes who are euthyroid. METHODS: A total of 111 people who were euthyroid underwent 75-g oral glucose tolerance tests, of whom 52 were assigned to the normal glucose tolerance and 59 to the prediabetes groups. Homeostatic model assessment of ß-cell function, insulinogenic index and areas under the curve for insulin and glucose were evaluated as indices of pancreatic ß-cell function. RESULTS: In both groups, BMI, fasting insulin, homeostasis model assessment ratio and HDL cholesterol correlated significantly with all indices of pancreatic ß-cell function. Free triiodothyronine correlated positively with all insulin secretion indices in the prediabetes group. Multiple linear regression analysis showed that free triiodothyronine was an independent variable that had a positive correlation with all indices of ß-cell function in the prediabetes group. By contrast, no such correlation was found in the normal glucose tolerance group. CONCLUSIONS: Free triiodothyronine is associated with both basal and glucose-stimulated insulin secretion in people with prediabetes who are euthyroid; therefore, the regulation of insulin secretion by thyroid hormones is a potentially novel therapeutic target for the treatment of diabetes.

Survival analysis of platinum-refractory patients with advanced esophageal cancer treated with docetaxel or best supportive care alone: a retrospective study.

The survival benefit of second-line chemotherapy with docetaxel in platinum-refractory patients with advanced esophageal cancer (AEC) remains unclear. A retrospective analysis of AEC patients with Eastern Cooperative Oncology Group performance status (PS)≤2 was performed, and major organ functions were preserved, who determined to receive docetaxel or best supportive care (BSC) alone after failure of platinum-based chemotherapy. The post-progression survival (PPS), defined as survival time after disease progression following platinum-based chemotherapy, was analyzed by multivariate Cox regression analysis using factors identified as significant in univariate analysis of various 20 characteristics (age, sex, PS, primary tumor location, etc) including Glasgow prognostic score (GPS), which is a well-known prognostic factor in many malignant tumors. Sixty-six and 45 patients were determined to receive docetaxel and BSC between January 2007 and December 2011, respectively. The median PPS was 5.4 months (95% confidence interval [CI] 4.8-6.0) in the docetaxel group and 3.3 months (95% CI 2.5-4.0) in the BSC group (hazard ratio [HR] 0.56, 95% CI 0.38-0.84, P=0.005). Univariate analysis revealed six significant factors: treatment, PS, GPS, number of metastatic organs, liver metastasis, and bone metastasis. Multivariate analysis including these significant factors revealed three independent prognostic factors: docetaxel treatment (HR 0.62, 95% CI 0.39-0.99, P=0.043), better GPS (HR 0.61, 95% CI 0.46-0.81, P=0.001), and no bone metastasis (HR 0.31, 95% CI 0.15-0.68, P=0.003). There was a trend for PPS in favor of the docetaxel group compared with patients who refused docetaxel treatment in the BSC group (adjusted HR 0.61, 95% CI 0.29-1.29, P=0.20). Docetaxel treatment may have prolonged survival in platinum-refractory patients with AEC.

Hepatic differentiation of mouse embryonic stem cells in a bioreactor using polyurethane/spheroid culture.

BACKGROUND: We have previously developed a hybrid artificial liver (HAL) using polyurethane foam (PUF)/hepatocyte spheroid culture. The PUF-HAL has been successfully scaled up to a clinical level. However, one of the most difficult problems for clinical application of HALs is obtaining a cell source. We now focused our attention on embryonic stem (ES) cells as a potential source for HAL. In this study, we investigated the differentiation of mouse ES (mES) cells into functional hepatocytes in the PUF-HAL module. METHODS: The PUF-HAL module included a cylindrical PUF block having many capillaries for medium flow. mES cells were immobilized in the module. To induce hepatic differentiation, growth factors were added to the culture medium. We evaluated cell density, gene expression analysis, and liver-specific functions. RESULTS: mES cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of PUF. mES cells proliferated by 20 days, achieving a high cell density (about 1 x 10(8) cells/cm3 PUF). Differentiating ES cells expressed endodermal-specific genes such as alpha-fetoprotein, albumin, and tryptophan 2, 3-deoxygenase. The activity of ammonia removal of mES cells per unit volume of the module was detectable by 15 days and increased with culture time. Maximal expression levels were comparable to those of primary (porcine and human) hepatocytes. SUMMARY: mES cells immobilized in the PUF module expressed liver-specific functions at high level, because of high cell density in culture and hepatic differentiation. These results indicated that PUF module-immobilized mES cells may be useful as a biocomponent of HALs.

Hepatic differentiation of embryonic stem cells in HF/organoid culture.

OBJECTIVE: The use of embryonic stem cells (ES cells) has recently received much attention as a novel cell source for various hybrid artificial organs. To use ES cells, it is necessary to be able to produce functional mature cells from ES cells in large quantities. We applied HF/organoid culture, where cultured cells formed cylindrical multicellular aggregates (organoids) in the lumen of hollow fibers, to mouse and cynomolgus monkey ES cells for hepatic differentiation. MATERIALS AND METHODS: ES cells were injected into hollow fibers. The hollow fibers were centrifuged to induce organoid formation and cultured in medium including factors for hepatic differentiation. To determine the characteristics of cells in the bundle, we evaluated gene expression and liver-specific functions. RESULTS: ES cells immobilized inside hollow fibers proliferated and formed cylindrical organoids. In mouse ES cell cultures, the expression of mRNAs of hepatocyte-specific genes increased with culture time. Ammonia removal activity detected at 15 days increased with culture time. Albumin secretion activity detected at 12 days increased by 21 days. In cynomolgus monkey ES cell cultures, ES cells showed spontaneous ammonia removal functions. The maximum levels of these functions per unit volume of the hollow fibers were roughly comparable to those of primary hepatocyte-organoids. CONCLUSIONS: ES cells differentiated into hepatocyte-like cells using the organoid culture technique. The results indicated that the combination of ES cells and an organoid culture technique was useful to obtain mature hepatocytes.

Development of articular cartilage grafts using organoid formation techniques.

In order to develop articular cartilage grafts, one must control shape and safety. We have developed scaffold-free culture methods in which the cells form multicellular aggregates (organoids). In this study, we applied the organoid culture method to chondrocytes attempting to reconstitute articular cartilage grafts. Primary rat costal chondrocytes and subcultured human articular chondrocytes were immobilized in hollow fibers by centrifugation at a density of 3 x 10(8) cells/cm3 to induce the formation of cylindrical-shaped organoids. To improve convenience, we developed a culture device to form sheet-shaped organoids (organoid-sheet). Primary bovine articular chondrocytes were cultured in this device. These organoids were evaluated by histological and gene expression analyses. In the primary rat culture system, chondrocytes formed cylindrical organoids in hollow fibers. Histochemical analysis revealed the presence of extracellular matrix (collagen and proteoglycan). The organoid maintained cartilage-specific gene expression (type II collagen, aggrecan) for 1 month of culture. In the subcultured human chondrocyte system, the organoid regained the decreased cartilage-specific gene expression. In the primary bovine culture system, the cells formed a 300 microm thickness organoid-sheet including abundant extracellular matrix. In conclusion, our organoid formation method was effective to form cartilage-like tissue. This result suggested that the technique may be applicable for the development of an articular cartilage graft.

Evaluation of a hybrid artificial liver module with liver lobule-like structure in rats with liver failure.

We studied the recovery of rats with fulminant hepatic failure (FHF) by treating them with our original hybrid artificial liver support system (HALSS). We developed an original artificial liver module having a liver lobule-like structure (LLS). This module consists of many hollow fibers regularly arranged in close proximity and hepatocyte aggregates (organoids) induced into the extra capillary space of the module by centrifugal force. The LLS module can express some liver specific functions at high levels and maintain them for several months in vitro. In this study, we evaluated the efficacy of our LLS-HALSS by using rats with FHF induced by a method that combined partial hepatectomy with hepatic ischemia. In the animal experiments, blood ammonia levels rapidly increased in the control group (sham-HALSS group). These rats died during or immediately after application of the sham-HALLS. On the other hand, in the LLS module application group (LLS-control group), the increase in blood ammonia was completely suppressed and all rats recovered. Blood constituents at 4 weeks after application were at normal levels, and the weight of the liver was the same as that of a normal rat. These results indicate that HALSS may be useful for treating liver failure patients until liver transplantation can be performed or until regeneration of the native liver occurs.

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer.

Using laser-captured microdissection and a real-time RT-PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P=0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P=0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55-3.67)), high DPD (HR: 2.04 (1.37-3.02)), low EGFR (HR: 0.34 (0.20-0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001-1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.

Application of chimerism-based drug-induced tolerance to rat into mouse xenotransplantation.

The current critical shortage of human donor organs has stimulated the feasibility of the xenogenic transplantation, such as swine to primate. We have previously reported the induction of donor-specific tolerance in MHC-disparated recipient mice by using our cyclophosphamide (CP)-induced tolerance conditioning. In this study, we examined the efficacy of our CP-induced tolerance conditioning in xenogenic transplantation model. F344 rats and B10 mice were used as donors and recipients. Recipient mice were treated with donor spleen cells, CP, Busulfan and bone marrow cells, with or without prior NK-cell depletion. Donor mixed chimerism, and the presence of donor reactive T-cell population were analysed by flow cytometry. The survival of the donor skin grafts were observed after the conditioning. Donor mixed chimerism was temporary induced but terminated at 10 weeks after treatments. Donor-specific prolongation of the skin graft survival was observed after the treatments, however, grafts were rejected in the long term. NK-cell depletion, prior to the treatments, did not affect the levels of the mixed chimerism or graft prolongation. The donor-reactive recipient T-cell population was remained the same level as the untreated mice, suggesting the failure of the induction of the central T-cell tolerance. Thus, partial efficacy of our CP-induced tolerance treatments in the rat to mice xenotransplantation was observed. Our results suggested that the additional treatments were required to establish the stable xenogenic tolerance.

Formation of a sheet-shaped organoid using rat primary hepatocytes for long-term maintenance of liver-specific functions.

In recent years, use of hepatocyte aggregates has led to development of a hybrid artificial liver support system (HALSS) that has high performance. However, in general, their thickness is 100 microm or more, and generation of a dead cell layer due to oxygen exhaustion inside the aggregates has been a universal problem. The present study proposes a novel organoid culture method with better performance than previous organoid culture methods by forming a sheet-shaped organoid (organoid-sheet) with a thickness of approximately 100 microm. The cell number of the organoid-sheet was maintained at approximately 75% of the initial number at 4 days of culture. On the other hand, that of a cylindrical organoid (cylindroid), which formed inside of a plasma separation hollow fiber with 285 microm inner diameter in our previous study, decreased to approximately 50% within 2 days. The ammonia removal rate of the cells in the organoid-sheet was higher than that of the cells in the cylindroid on the first day, but it decreased during the culture time. At day 15, the rate was reduced by almost 50% with respect to the value on the first day. The cells in the cylindroid displayed a lower ammonia removal rate. A significant difference was not observed between the albumin synthesis rates of the two cultures on the first day. However, over a period of time the cells in the organoid-sheet showed a higher albumin synthesis rate than cells in the cylindroid. As this novel organoid maintains these functions for at least 1 month, it is expected to be applied for the development of a HALSS with higher performance.

Efficacy and limitations of cyclophosphamide-induced tolerance against alphaGal antigen.

In the present study, we have elucidated the efficacy of two cyclophosphamide (CP)-induced tolerance protocols for the induction of B-cell tolerance against Galalpha1-3Galbeta1-4GlcNAc (alphaGal) antigens. alpha1,3-galactosyltransferase-deficient (GalT-/-; H-2(b/d)) mice received with 1 x 10(8) AKR (alphaGal+/+ H-2k) spleen cells (SC) followed by 200 mg/kg CP, or alternatively followed by 200 mg/kg CP, 30 mg/kg Busulfan (BU) and 1 x 10(8) T-cell-depleted AKR bone marrow cells (BMC). The generation of both anti-alphaGal and anti-donor antibodies were completely suppressed, but normal antibody production against third party antigens was observed after BALB/c skin grafting in both groups of GalT-/- mice. In GalT-/- mice, treated with SC and CP, mixed chimerism was not observed. Cellular rejection was observed in grafted donor AKR hearts with an absence of humoral rejection, whereas humoral rejection was observed in untreated GalT-/- mice. On the other hand, long-term mixed chimerism and permanent acceptance of donor AKR skin graft and heart graft were achieved in GalT-/- mice treated with SC, CP, BU and BMC. These results demonstrate the efficacy of classical drug-induced tolerance in the induction of B-cell tolerance against alphaGal antigens. However, induction of stable mixed chimerism was required for the suppression of cellular rejection.

Differentiation effects by the combination of spheroid formation and sodium butyrate treatment in human hepatoblastoma cell line (Hep G2): a possible cell source for hybrid artificial liver.

The aim of this study was to investigate the feasibility of human hepatoblastoma cell line (Hep G2), which differentiates by spheroid formation, and treatment with sodium butyrate (SB) as a cell source for hybrid artificial liver (HAL). Hep G2 spontaneously formed spheroids in polyurethane foam (PUF) within 3 days of culture and restored weak ammonia removal activity. Treatment with SB, which is a histone deacetylase inhibitor, further increased the ammonia removal activity of Hep G2 spheroids in a concentration-dependent manner. The activation of ornithine transcarbamylase--a urea cycle enzyme--was significantly related to the upregulation of ammonia removal by spheroid formation, but scarcely contributed to the further upregulation following SB treatment. In contrast with ammonia removal, treatment with SB reduced the albumin secretion of Hep G2 spheroids in a concentration-dependent manner. In the PUF-HAL module in a circulation culture, the ammonia removal rate and albumin secretion rate (per unit volume of the module) of Hep G2 spheroids treated with 5 mM SB were almost the same as those of primary porcine hepatocyte spheroids. These results suggest that simultaneous use of spheroid formation and SB treatment in Hep G2 is beneficial in enhancing the functions of human hepatocytes with potential applications in regenerative medicine and drug screening.

Hepatocyte organoid culture in elliptic hollow fibers to develop a hybrid artificial liver.

A novel organoid culture was developed in which hepatocytes maintain high liver functions for more than several weeks in vitro. The main disadvantage of tissue-engineered organoids is the lack of a blood vessel structure between the aggregated cells. Because of depletion of oxygen, the thickness from the surface of an organoid at which hepatocytes can survive is limited. This study showed that a rat hepatocyte organoid that forms by using centrifugal force in a hollow fiber (HF) had a survival limit thickness of about 80 - 100 microm from the surface of the organoid. Based on the value, we designed an elliptic HF having less than 150 microm minor diameter by using a simple annealing method. All hepatocytes were supplied with oxygen and formed an organoid without a dead cell layer in this HF A hepatocyte organoid in an elliptic HF maintained ammonia removal activity twice as high as in the original HF for at least one month during culture. Albumin secretion activity of an organoid in an elliptic HF was also maintained for at least one month and was the same level as that of liver in a living body. In conclusion, organoid culture by using an elliptic HF seems to be a promising technique to develop a hybrid artificial liver.

Effects of N-alpha-methyl-histamine on human H(2) receptors expressed in CHO cells.

BACKGROUND: Production of N-alpha-methyl-histamine (NAMH), a histamine H(3) receptor (H3R) agonist, is reportedly promoted in Helicobacter pylori infected human gastric mucosa. NAMH was suggested to act directly on histamine H(2) receptors (H2Rs) in animals to stimulate acid secretion and to be a H2R agonist. As H2Rs and H3Rs play different roles in gastric acid secretion, it is very important to verify that NAMH is a H2R agonist. AIMS: To determine whether NAMH is a H2R agonist, as well as a H3R agonist. METHODS: We used a Chinese hamster ovary (CHO) cell line expressing human H2Rs (CHO-H2R) and control CHO cells. Expression of human H2Rs was confirmed by tiotidine binding. cAMP production in CHO-H2R and control cells in response to histamine or NAMH was measured. cAMP production in response to 10(-7) M NAMH was also measured in the presence or absence of the H2R antagonist famotidine and the H3R antagonist thioperamide. RESULTS: NAMH dose dependently stimulated cAMP productions in CHO-H2R cells. This production was inhibited by famotidine but not by thioperamide. Control CHO cells were unresponsive to either histamine or NAMH. In addition, the effect of NAMH, in terms of cAMP production in CHO-H2R cells, was more potent than that of histamine-that is, with a lower EC(50) concentration and higher maximal cAMP production. Both NAMH and histamine, but not R-alpha-methyl-histamine, effectively inhibited [(3)H] tiotidine binding to CHO-H2R cells. CONCLUSIONS: NAMH, which is produced in the gastric mucosa by H pylori, is a potent H2R agonist as well as a H3R agonist.

Recovery of rats with fulminant hepatic failure by using a hybrid artificial liver support system with polyurethane foam/rat hepatocyte spheroids.

We studied the recovery of rats with fulminant hepatic failure (FHF) by treating them with our original hybrid artificial liver support system (HALSS). FHF was induced by a two-thirds partial hepatectomy and 10 minutes of hepatic ischemia. Rats with FHF were treated with a polyurethane foam/spheroid HALSS including 2.0 x 10(8) hepatocytes for 1 hour (HALSS group, n = 5), and with the same system without hepatocytes in the artificial liver module as a control experiment (sham-HALSS group, n = 3). The level of blood constituents, ammonia, glucose and creatinine, showed no major difference between the two groups at the end of treatment. All rats in the sham-HALSS group died within 5 hours after treatment. However, the level of blood constituents of rats with FHF in the HALSS group improved with time, and all rats in the HALSS group recovered. Liver tissue of rats treated with HALSS showed cell mitosis and improvement from injury. These results indicated that our HALSS has a strong possibility to induce recovery from hepatic failure.

Allelic losses as prognostic markers for breast cancers.

Specific allelic losses in the DNA of tumor cells are potential indicators of postoperative prognosis. Patients whose tumors showed allelic losses at 1p34, 3p25, 8p22, 13q12, 17p13.3, or 17q21.1 had a significantly higher risk of postoperative mortality than women whose tumors retained both alleles at those loci (the 5-year mortality rates in patients with loss vs those with retention were: at 1p34, 23% vs 10%, P = 0.0100; at 3p25, 22% vs 9%, P = 0.0014; at 8p22, 24% vs 7%, P = 0.0177; at 13q12, 19% vs 8%, P = 0.0093; at 17p13.3, 19% vs 9%, P = 0.0078; and at 17q21.1, 17% vs 10%, P = 0.0475). Allelic losses at these loci may serve as negative prognostic indicators to guide postoperative management, especially in the selection of patients who should be offered intensive adjuvant therapy.

Association of allelic losses at 3p25.1, 13q12, or 17p13.3 with poor prognosis in breast cancers with lymph node metastasis.

To identify specific allelic losses that might correlate with postoperative mortality of patients with node-positive breast carcinomas, we examined tumors from a cohort of 263 such patients, who were followed clinically for 5 years postoperatively, for allelic losses among 18 microsatellite markers. Patients whose tumors had lost an allele at 3p25.1, 13q12, or 17p13.3 had significantly higher risks of mortality than those whose tumors retained both alleles at those loci. At 3p25.1, the 5-year mortality rate was 33.8% among patients with losses vs. 16.8% with retention (P = 0.0154); at 13q12, 30.3% vs. 13.0% (P = 0.0241); and at 17p13.3, 30.4% vs. 16.2% (P = 0.0243). Combined losses at 3p25.1 and 17p13.3 increased the predicted postoperative mortality risk by a factor of 4.9 (5-year mortality rate of 38.2% vs. 8.0%, P = 0.0006), and combined losses at 3p25.1 and 13q12 raised the predicted postoperative mortality risks by a factor of 2.9 (34.7% vs. 12.7%, P = 0.0441). These data indicate that loss of heterozygosity (LOH) at any one or a pair of loci at 3p25.1, 13q12, or 17p13.3 is a significant predictor of postoperative mortality for breast-cancer patients.

Study on endoscopic esophageal mucosal resection with ligating device. I--Clinical study.

BACKGROUND/AIMS: EEMRL (endoscopic esophageal mucosal resection with a ligating device) has become increasingly popular. In this article, we review 13 clinical cases of EEMRL. METHODOLOGY: Since 1993, we have performed EEMRL to treat 15 lesions in 13 patients. Twelve squamous cell carcinomas (mucosal cancer in 10 and submucosal cancer in 2) were included among the 15 lesions. RESULTS: EEMRL failed to achieve complete resection of the 2 submucosal lesions (3.0 and 2.8 cm in maximum diameter). However, esophageal lesions could be removed successfully when 2.5 cm or less in maximum diameter. The procedure was not associated with any complication. CONCLUSIONS: Our clinical study showed that this technique may be indicated for esophageal cancer with a maximum diameter < or = 2.5 cm and confined to the mucosa. EEMRL is a technically easy and minimally invasive therapy which could be useful for the treatment of early esophageal cancer.

Study on endoscopic esophageal mucosal resection with ligating device. II--Experimental study.

BACKGROUND/AIMS: This study reports on animal experiments regarding the safety of endoscopic esophageal mucosal resection with a ligating device (EEMRL), as well as the amount of mucosa which can be removed by this technique, the depth of resection and the feasibility of piecemeal resection. METHODOLOGY: Three experiments were performed in six mongrel dogs under general anesthesia. RESULTS: When EEMRL was done without submucosal injection of saline, resection reached the muscular layer and caused esophageal perforation. The average dimensions of the mucosal pieces resected using 8-, 10-, and 12-mm devices was 13 x 10 mm, 18 x 15 mm, and 22 x 18 mm, respectively. Resection reached the mid-plane of the submucosa and the depth was almost uniform. After piecemeal resection, there was no macroscopically visible mucosa at the resection site and each mucosal piece was resected along the mid-plane of the submucosa. CONCLUSIONS: The experimental study indicated that submucosal injection of saline is essential to prevent esophageal perforation. It also showed that EEMRL allows resection up to the mid-plane of the submucosa, that the 12-mm device allows en bloc resection of lesions < or = 15 mm in diameter and that EEMRL is suitable for piecemeal resection.