Pseudoileus caused by primary visceral myopathy in a Han Chinese patient with a rare MYH11 mutation: A case report.

BACKGROUND: Chronic intestinal pseudo-obstruction (CIPO) is a syndrome of intestinal motor dysfunction caused by intestinal nerve, muscle, and/or Cajal stromal cell lesions. CIPO is a serious category of gastrointestinal dynamic dysfunction, which can eventually lead to the death of patients with intestinal failure. Due to considerable phenotypic heterogeneity, the estimated incidence of CIPO is 1/476190 and 1/416666 in men and women, respectively. According to the etiology, CIPO can be divided into idiopathic and secondary, of which the latter is the most common, often secondary to tumor, virus infection, connective tissue disease, neurological diseases, and endocrine diseases. Idiopathic CIPO in the intestinal tract is divided into visceral myopathy, neuropathy, and stromal cell lesions according to the location. Surgery is usually not recommended for CIPO, because it often does not benefit patients with CIPO, and postoperative intestinal obstruction is likely to occur, which may even worsen the condition. CASE SUMMARY: Here, we describe the case of a 43-year-old male Han Chinese patient with a 15-year history of recurrent abdominal distention with no clear cause. The results of physical, biochemical, and other relevant examinations showed no clear abnormalities. Contrast-enhanced computed tomography (CT) indicated a large duodenum, clear expansion of the intestinal lumen, and CIPO. Whole exome sequencing (WES) of the patient and his mother confirmed the diagnosis of primary familial visceral myopathy type 2 chronic pseudoileus with a rare heterozygous gene mutation in MYH11. This is the second reported case of CIPO with a heterozygous MYH11 [NM_001040113.1: c.5819delC (p.Pro1940Hisfs*91)] mutation. CONCLUSION: This case report indicates that physicians can perform routine clinical examinations, CT, and WES to achieve a diagnosis and treatment of CIPO in early disease stages.

Prevalence of colorectal neoplasm in Chinese patients with high-risk coronary artery disease classified by the Asia-Pacific Colorectal Screening score.

OBJECTIVE: The aim of this study was to investigate the prevalence of colorectal neoplasms in patients coronary artery disease (CAD) with or without a family history of colorectal cancer (CRC). METHODS: In this cross-sectional study, individuals with suspected CAD in the absence of cancer-related symptoms underwent coronary angiography for the first time, and were divided into CAD and non-CAD groups. Colonoscopy was performed in individuals at high-risk tier based on their Asia-Pacific colorectal screening (APCS) score. Their waist circumference (WC), height and body weight were measured. RESULTS: There were 634 of 1157 individuals at a high risk of developing advanced colorectal neoplasms, 91.0% (577/634) of whom were male smokers. The proportion of CAD patients in the high-risk tier was 81.5% (517/634), while the prevalences of adenomas (32.1% vs 22.2%, P < 0.05) and advanced adenomas (14.7% vs 8.5%, P < 0.05) were significantly higher in the CAD group than in the non-CAD group. After 83 individuals with a family history of CRC were excluded, only the prevalence of adenomas was still significantly higher in the CAD group than in the non-CAD group (25.5% vs 16.0%, P < 0.01). Body mass index (BMI) ≥ 25 kg/m(2) was correlated with the occurrence of adenomas (OR 2.133, 95% CI 1.219-3.730, P = 0.008) in CAD patients. CONCLUSIONS: Even in the absence of family history of CRC, CAD patients at a high risk of developing advanced colorectal neoplasms classified by the APCS score still showed a remarkably high prevalence of colorectal adenomas. Moreover, the association between the occurrence of adenomas and CAD was stronger in overweight (BMI ≥ 25 kg/m(2)) individuals.

Low-dose azathioprine effectively improves mucosal healing in Chinese patients with small bowel Crohn's disease.

OBJECTIVE: To evaluate mucosal healing after 24-month low-dose azathioprine (AZA) treatment in Chinese patients with moderate small bowel Crohn's disease (CD). METHODS: Patients with lesions mainly located at the ileum were screened by baseline multislice computed tomography enterography and anal-route double-balloon enteroscopy (DBE). They were naive to any immunomodulators and biological agents before the enrollment. Lesions from 150 cm of the terminal ileum proximal to ileocecal valve were assessed by DBE with the simple endoscopic score for CD (SES-CD) after 12 and 24 months of low-dose AZA treatment, respectively. RESULTS: The average maximal tolerance dose of AZA was 61.8 ± 17.2 mg/day. The total rates of complete, near-complete, partial and no mucosal healing in 36 patients were 19.4%, 5.6%, 27.8% and 47.2% at month 12 and 30.6%, 25.0%, 33.3% and 11.1% at month 24, respectively. The baseline SES-CD (odds ratio [OR] 2.71, 95% confidence interval [CI] 1.11-6.63, P = 0.029) and duration of disease (OR 1.27, 95% CI 1.10-1.47, P = 0.001) were two relevant factors associated with the mucosal healing of patients with small bowel CD. CONCLUSION: A 24-month low-dose AZA regimen as maintenance treatment in moderate small bowel CD could achieve a higher mucosal healing rate than that of 12-month treatment in Chinese patients, especially in those with duration of disease less than 12 months and a baseline SES-CD of 5 or 6.

Gallbladder gallstone disease is associated with newly diagnosed coronary artery atherosclerotic disease: a cross-sectional study.

BACKGROUND AND AIMS: The association between gallstone disease and coronary artery atherosclerotic disease (CAD) remains unclear. To clarify their relationship, patients with CAD newly diagnosed by coronary angiography were investigated in this cross-sectional study. METHODS: The study cohort consisted of 1,270 patients undergoing coronary angiography for the first time between January 2007 and September 2011. Patients with ≥50% diameter stenosis in any major coronary artery on coronary angiography were defined as being CAD positive (n = 766) and those with no stenosis as CAD negative (n = 504). Multivariate logistic regression was used to investigate the relationship between gallstone disease and CAD. The odds ratios (OR) of factors associated with CAD were calculated. In addition, CAD-positive and CAD-negative patients were matched one-to-one by age, gender and metabolic syndrome (MetS), and the association between gallbladder disease and CAD was determined. RESULTS: The prevalence of gallstone disease was significantly higher in CAD-positive than in CAD negative patients (149/766 [19.5%] vs 57/504 [11.3%], P<0.01). Gallstone disease was significantly associated with CAD (adjusted OR = 1.59, 95% confidence interval [CI] 1.10-2.31). Following matched pairing of 320 patients per group, gallstone disease remained significantly associated with CAD (adjusted OR = 1.69, 95% CI: 1.08-2.65). CONCLUSION: Gallstone disease is strongly associated with CAD diagnosed by coronary angiography.

Fatty liver index correlates with non-alcoholic fatty liver disease, but not with newly diagnosed coronary artery atherosclerotic disease in Chinese patients.

BACKGROUND: Fatty liver index (FLI) was recently established to predict non-alcoholic fatty liver disease (NAFLD) in general population, which is known to be associated with coronary artery atherosclerotic disease (CAD).This study aims to investigate whether FLI correlates with NAFLD and with newly diagnosed CAD in a special Chinese population who underwent coronary angiography. METHODS: Patients with CAD (n = 231) and without CAD (n = 482) as confirmed by coronary angiography were included. Among them, 574 patients underwent B-ultrosonography were divided into NAFLD group (n = 209) and non-NAFLD group (n = 365). Correlation between FLI and NAFLD was analyzed using pearson's correlation. The associations between FLI and NAFLD as well as CAD were assessed using logistic regression. The predictive accuracy of FLI for NAFLD was evaluated using receiver operating characteristics (ROC) curve analysis. RESULTS: FLI was significantly higher in NAFLD group (37.10 ± 1.95) than in non-NAFLD group (17.70 ± 1.04), P < 0.01. FLI correlated with NAFLD (r = 0.372, P < 0.001). The algorithm for FLI had a ROC-AUC of 0.721 (95% CI: 0.678-0.764) in the prediction of NAFLD. Logistic regression analysis showed that FLI was associated with NAFLD (adjusted OR = 1.038, 95% CI: 1.029-1.047, P < 0.01). The proportion of patients with CAD did not differ among the groups of FLI ≤ 30 (32.3%), 30-60 (31.0%), and ≥60 (35.3%). No significant association was found between FLI and CAD (adjusted OR = 0.992, 95% CI: 0.981-1.003 in men and OR = 0.987, 95% CI: 0.963-1.012 in women, P > 0.05). CONCLUSIONS: FLI showed good correlation with NAFLD in patients who underwent coronary angiography, but not with newly diagnosed CAD. This might be underestimated because some patients in non-CAD group may have other underlying cardiovascular diseases.

Prognostic factors and outcome of resected patients with gastrointestinal stromal tumors of small intestine.

We document the clinical behavior of gastrointestinal stromal tumors (GISTs) of the small intestine and identify predictors for long-term disease-free survival (DFS) for small intestine GIST patients. From December 2001 to 2008, 114 consecutive patients with mesenchymal tumors involving the small intestine were enrolled. There were 54 male and 60 female (50.6%) patients. After a median follow-up period of 36 months (ranging from 12 to 96 months), recurrence was noted in 19 patients (16.7%) with a median time of 20 months (ranging from 7 to 50 months). There were 12 patients (10.5%) who died of GISTs with a median time from recurrence to death of 14 months (ranging from 8 to 22 months). Univariate analysis by log-rank test indicated that tumor size and mitotic activity were statistically significant for DFS (P = 0.001 and 0.036, respectively). Tumor size was the only significant predictive factor for DFS according to multivariate analysis (P = 0.006). Small tumor size, indicating low risk, predicted more favorable DFS of small intestine GIST patients who underwent curative resection.

Stat3 is involved in angiotensin II-induced expression of MMP2 in gastric cancer cells.

The expression of matrix metalloproteinase-2 (MMP2) has been linked with tumor invasion, angiogenesis, and metastasis. It has been reported that angiotensin II (Ang II) can induce MMP2 expression in gastric cancer cells. However, the molecular basis for Ang II regulates MMP2 expression in gastric cancer cells remains unclear. The aim of our study is to explore whether angiotensin II could induce MMP2 expression mediated through the Stat signaling pathway and its potential mechanism. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-binding assays was employed to determine the DNA-STAT binding activity. MMP2 and VEGF expression was analyzed with real-time PCR and Western blots. To examine the role of Stat3 in angiotensin II-induced MMP2 expression, A JAK-specific inhibitor and AG490 were used. Angiotensin II activated STAT-DNA binding activity in dose-dependent manners in gastric cancer cells. AG490 markedly inhibited angiotensin II-induced Stat3 activation and the expression of MMP2 and VEGF in gastric cancer cells. These results indicate that Stat3 may at least in part mediate angiotensin II-induced MMP2 mRNA expression in human gastric cancer cells. The activation of the JAK/Stat3 signaling pathway plays an important role in the progression of gastric cancer and that blockade of JAK/Stat3 signals may provide a novel therapeutic strategy for gastric cancer.

Suppression of gastric cancer growth by baculovirus vector-mediated transfer of normal epithelial cell specific-1 gene.

AIM: To study the inhibitory effect of baculovirus-mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.

Angiotensin II type 1 receptor antagonist suppress angiogenesis and growth of gastric cancer xenografts.

Angiotensin II (Ang II) has been reported to promote tumor progression, tumor growth and angiogenesis in many cancers. We previously observed that angiotensin II type 1 receptors (AT1R) were upregulated in human gastric cancer and may be involved in the progression of gastric cancer. We studied the effects of AT1R antagonist on angiogenesis and growth in gastric cancer xenografts to observe the mechanism action of AT1R in the gastric cancer. The results showed that the growth of gastric cancer cells was significantly suppressed by treatment with AT1R antagonist. In vivo, TCV-116, at doses of both 2 and 5 mg/kg/day, significantly suppressed tumor growth in mice (47.3 and 70.2%). Microvessel density was significantly decreased by TCV-116 (3.4 +/- 0.9 and 2.8 +/- 0.5 per field) compared with the control group (12.9 +/- 1.1 per field), and VEGF expression was significantly suppressed by AT1R antagonist. These results demonstrate that AT1R plays an important role in the progression of gastric cancer. Suppression tumor angiogenesis could be one of the mechanisms by which AT1R antagonist suppresses the growth of gastric cancer. These findings also provide a theoretical basis for the future clinical application of AT1R antagonist against gastric cancer.

Angiotensin II type 1 receptor expression in human gastric cancer and induces MMP2 and MMP9 expression in MKN-28 cells.

Angiotensin II (Ang II), a main effector peptide in the renin-angiotensin system, acts as a growth-promoting and angiogenic factor via angiotensin II receptor1 (AT1R). In this study, we investigated the expression of angiotensin II type1 receptor (AT1R) in gastric cancer and the effects of Ang II on the expression of MMP2 and MMP9 in the human gastric cancer cell line MKN-28 cells. The expression of the Ang II type I receptor was examined by western and immunocytochemistry in gastric cancer cell lines and detected by real-time PCR and immunohistochemistry in normal and gastric cancer tissues. The expression of MMP2 and MMP9 were detected by real-time PCR and western after treatment with Ang II and/or AT1R antagonist. AT1R were expressed in all human gastric cancer lines and the expression of AT1R was significantly higher in cancer tissues than that in normal gastric tissues (P < 0.01). Furthermore, Ang II promoted the expression of MMP2 and MMP9 in MKN-28 cells, and the stimulatory effects of Ang II could be blocked by AT1R antagonist. These results suggest that AT1R is involved in the progression of gastric cancer and may promote the angiogenesis of gastric cancer cell line (MKN-28), and these effects may be associated with the upregulation of MMP2 and MMP9.

XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer.

BACKGROUND: XIAP-associated factor 1 (XAF1) negatively regulates the function of the X-linked inhibitor of apoptosis protein (XIAP), a member of the IAP family that exerts antiapoptotic effects. The extracellular signal-regulated kinase (ERK) pathway is thought to increase cell proliferation and to protect cells from apoptosis. The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer. METHODS: Four human colon cancer cell lines, HCT1116 and Lovo (wildtype p53), DLD1 and SW1116 (mutant p53), were used. Lovo stable transfectants with XAF1 sense and antisense were established. The effects of dominant-negative MEK1 (DN-MEK1) and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined. The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay. Cell proliferation was measured by MTT assay. Apoptosis was determined by Hoechst 33258 staining. RESULTS: U0126 increased the expression of XAF1 in a time- and dose-dependent manner. A similar result was obtained in cells transfected with DN-MEK1 treatment. Conversely, the expression of XIAP was down-regulated. Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection. rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression. Overexpression of XAF1 was more sensitive to U0126-induced apoptosis, whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation. CONCLUSIONS: XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation, which required de novo protein synthesis. The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Downregulation and CpG island hypermethylation of NES1/hK10 gene in the pathogenesis of human gastric cancer.

The normal epithelial cell-specific-1 (NES1)/Kallikrein 10 gene is proposed to be a novel putative tumor suppressor gene in several malignant diseases. The role of NES1 gene in gastric cancer has not been fully understood. Our study revealed that CpG island hypermethylation plays an important role in the downregulation of NES1 mRNA expression in gastric cancer. In situ hybridization showed that loss or reduction of NES1 mRNA expression is associated with differentiation level during tumor progression suggesting that NES1 inactivation might contribute to the malignant progression of human gastric cancers.

Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers.

BACKGROUND & AIMS: X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression. METHODS: XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis. RESULTS: Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers. CONCLUSIONS: A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at -2nd, -1st, and +3rd positions are functionally more important in its transcriptional regulation.

Induction of apoptosis and cell cycle arrest by a specific c-Jun NH2-terminal kinase (JNK) inhibitor, SP-600125, in gastrointestinal cancers.

The c-Jun NH(2)-terminal kinase (JNK) is activated in several tumor cell lines. The aim of this study was to determine the effects of SP-600125, a specific JNK inhibitor, on the viability, apoptosis, cell cycle distribution of gastrointestinal cancer cells, and the potential anti-tumor mechanisms. Three gastric cancer cell lines, AGS, BCG-823 and MKN-45, and three colorectal cancer cell lines, SW1116, COLO205 and HT-29, were used. Cells were treated with SP-600125, and cell viability, apoptosis and cell cycle distribution, caspase-3 activity, expression of JNK and apoptosis related proteins were detected. SP-600125 inhibited cell proliferation by 10-80% for the different cell lines, and increased apoptosis by 1.5-4.5 folds for COLO205, BCG-823, MKN-45, AGS cells. Caspase-8 and caspase-3 were involved in the induction of apoptosis. SP-600125 caused G2/M cell cycle arrest and elevation of cyclin B1 and p27(kip). The differential response in cells to SP-600125 was associated with the basal level of phosphorylated JNK2. It is concluded that SP-600125 inhibits proliferation, induces apoptosis and causes cell cycle arrest in gastrointestinal cancer cells, indicating that JNK inhibitors have an anti-tumor effect and are potential therapeutic agents for cancers.

Activation of STAT3 signaling in human stomach adenocarcinoma drug-resistant cell line and its relationship with expression of vascular endothelial growth factor.

AIM: To investigate the difference in activation of STAT3 signaling between two human stomach adenocarcinoma cell lines: 5-fluorouracil resistant cell line and its parental cell line, and to evaluate its relationship with the expression of vascular endothelial growth factor (VEGF). METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were used to detect the expression of phospho-STAT3 protein and constitutive activation of STAT3 in two human stomach adenocarcinoma cell lines, 5-fluorouracil resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of VEGF was analysed by semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry. RESULTS: The expressions of phospho-STAT3 protein and constitutive activation of STAT3 between two human stomach adenocarcinoma cell lines were different. Compared with the parental cell line SGC7901, the STAT3-DNA binding activity and the expressive intensity of phospho-STAT3 protein were lower in the drug-resistant cell line SGC7901/R. The expression levels of VEGF mRNA and its encoded protein were also decreased in drug-resistant cell line. CONCLUSION: Over-expression of VEGF may be correlated with elevated STAT3 activation in parental cell line. Lower VEGF expression may be correlated with decreased STAT3 activation in resistant cell line, which may have resulted from negative feedback regulation of STAT signaling.

[Constitutive activation and clinical significance of Stat3 in human gastric cancer tissues and cell lines].

OBJECTIVE: To investigate the activation of signal transducers and activators of transcription 3 (Stat3) in different types of gastric cancer cell lines and tissues and evaluate the relationship with their clinicopathological parameters. METHODS: Western blotting and electrophoretic mobility shift assay (EMSA) were used to detected the expression of Stat3 protein and Stat3 DNA-binding activity in normal human gastric epithelial cell line 3T3 and five gastric cancer cell lines with different differentiation: MKN28, SGC7901, MKN45, AGS and NCI-SNU-1, respectively. The localization of phospho-Stat3 was determined by immunocytochemistry. The expressive intensity of phospho-Stat3 protein in 50 cases of gastric cancer tissues and adjacent normal mucosa were measured by immunohistochemistry. RESULTS: Compared with normal gastric epithelial cell line 3T3, elevated activities of Stat3 were found in five different human gastric cancer cell lines. The Stat3 DNA-binding activity in moderately and poorly differentiated stomach adenocarcinoma cell lines (SGC7901, MKN45 and AGS) was higher than that of other cell lines (MKN28 and NCI-SNU-1). Phospho-Stat3 was detected primarily in the nuclei of AGS cells. The expressive intensity of phospho-Stat3 protein was significantly increased in gastric cancer tissues as compared with the adjacent normal gastric mucosa, especially in moderately and poorly differentiated cancers (both P < 0.05). The expressive intensity of phospho-Stat3 protein in stage II and stage III tumors was higher than that in stage I tumors (P < 0.05). No statistic difference of phospho-Stat3 expression was found between stage IV and stage I tumors (P > 0.05). The expression of phospho-Stat3 was closely correlated with the differentiation of gastric cancer. CONCLUSION: Elevated activity of Stat3 can be found in different types of human gastric cancer cell lines and gastric cancer. JAK/STAT signal transduction pathway may play an important role in the development of human gastric cancer.

[The impact of decreased Stat3 activation on 5-fluorouracil resistance of human gastric cancer cell line].

OBJECTIVE: To investigate the relationship between different activation of Stat3 signaling and the drug resistance mechanisms in two human gastric cancer cell lines, 5-fluorouracil (5-FU) resistant cell line and its parental cell line. METHODS: Electrophoretic mobility shift assay and Western blot were used to detected Stat3 DNA-binding activity and the expression of phospho-Stat3 protein in 5-FU resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of Stat3 and vascular endothelial growth factor (VEGF) were analysed with semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry. RESULTS: The constitutive activation of Stat3 and the expression of phospho-Stat3 protein were different in two human gastric cancer cell lines. Compared with the parental cell line SGC7901, the Stat3-DNA binding activity and the expressive intensity of phospho-Stat3 protein were lower in the drug-resistant cell line SGC7901/R. The expression level of Stat3 mRNA was also decreased in drug resistant cell line, so did VEGF mRNA and its encoded protein. CONCLUSIONS: The decreased Stat3 activation in 5-FU resistant human gastric cancer cell line SGC7901/R is related to the drug resistance mechanisms and may be correlated with the lower VEGF expression.

[An improved quantitative method for evaluation of ischemic injury and neuroprotection in mouse brain slices].

OBJECTIVE: To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices. METHODS: An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects. RESULT: The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect. CONCLUSION: The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.