Changes in tumor necrosis factor alpha and myeloper-oxidase in mouse models of local cerebral infarction induced by photochemical method

BACKGROUND: Lots of evidences have demonstrated that acute inflammatory reaction plays an important role in cerebral ischemia and cerebral ischemia/reperfusion injury. Tumor necrosis factor (TNF), as one of important inflammatory cytokines, also participates in the injury. OBJECTIVE: To observe the changes in TNF-α expression and myeloperoxidase (MPO) activity of mouse models of local cerebral infarction induced by photochemical method, and analyze the correlation of TNF-α expression and MPO activity. DESIGN: Randomized controlled experiment. SETTING: Laboratory of Cerebral Microcirculation, Taishan Medical College.MATERIALS: Sixty involved male adult Kunming mice were provided by the Experimental Animal Center of Shandong University. TNF-α primary antibody, kits for enzyme-linked immunosorbent assay(ELISA) and streptavidin-biotin complex immunohistochemical dyeing kit were purchased from Boster Company(Wuhan). MPO kit was purchased from Jiancheng Bioengineering Institute (Nanjing). Cold light source was developed by Hengfa Co.,Ltd.( LG-150, Xuzhou). METHODS: This experiment was carried out in the Laboratory of Cerebral Microcirculation of Taishan Medical College between July 2004 and July 2005. The involved 60 Kunming mice were randomized into 3 groups: normal control group (n =6), sham-operation group (n =6) and model group (n =48). Mice in the model group were observed at 30 minutes, 1, 3, 6, 12, 24, 48 and 72 hours after illumination, separately, 6 mice at each time point. In the model group, mice models of local cerebral infarction were developed as follows: The mice were anesthetized to expose left skulls. Taking 2 mm left to sagittal suture and 2 mm posterior to coronal suture as center, a field with diameter of 3 mm for illumination was set. The optical fiber detecting head of cold light source was vertically close to exposed skull. The mice were injected with rose Bengal for 5 minutes, and then cold light source was open for 10 minutes. Illumination was omitted in the sham-operation group. Mice in the control group were not modeled. At postoperative 6 hours, TNF-α expression in infracted-side cortex was detected with immunohistochemical method and ELISA, and MPO activity in infracted-side cortex with chromatometry. MPO activity could reflect the infiltration degree of neutrophils in tissue. Stronger activity indicated severer infiltration. Single-factor analysis of variance was used for comparison among groups, q test for pairwise comparison and correlative analysis for detecting the inter-parameter correlation. MAIN OUTCOME MEASURES: Changes in TNF-α expression and MPO activity of left cortex of mice in each group. RESULTS: Sixty mice were involved in the final analysis. After cerebral infarction, TNF-α positive cells were neurons and glial cells mainly, distributing in and around the infarct region. TNF-α expression in cortex of mice of sham-operation group was （615.7±16.1） ng/L, and that of model group increased to （792.2±17.8） ng/L at 3 hours after illumination, and reached peak [（921.9±23.9） ng/L] at 6 hours after illumination, and decreased to （848.0±30.6） ng/L at 12 hours after illumination and recovered to the normal level [（625.3±14.3） ng/L] at 72 hours after illumination. MPO activity of sham-operation group was （7.151±0.433） nkat/g, and that of model group increased to （10.469±0.600） nkat/g at 3 hours after illumination, reached the peak [（15.486±0.650） nkat/g] at 12 hours after illumination, decreased to （11.052±0.617） nkat/g at 24 hours after illumination and recovered to the normal level [（7.418±0.617） nkat/g] at 72 hours after illumination. Change of MPO activity lagged behind that of TNF-α, and correlative analysis showed that the both were positively correlated（r =0.953, P < 0.01）.CONCLUSION: In the acute stage of cerebral infarction of mice induced by photochemical method, TNF-α expression in infarcted-side cortex is closely related with infiltration of neutrophils. TNF-α induces inflammatory cells to intrude into ischemic brain tissue, and participates in the inflammatory reaction process at the early stage of cerebral ischemia.

Changes in tumor necrosis factor alpha and myeloper-oxidase in mouse models of local cerebral infarction induced by photochemical method

BackgroundLots of evidences have demonstrated that acute inflammatory reaction plays an important role in cerebral ischemia and cerebral ischemia/reperfusion injury. Tumor necrosis factor (TNF), as one of important inflammatory cytokines, also participates in the injury.ObjectiveTo observe the changes in TNF-α expression and myeloperoxidase (MPO) activity of mouse models of local cerebral infarction induced by photochemical method, and analyze the correlation of TNF-α expression and MPO activity.DesignRandomized controlled experiment.SettingLaboratory of Cerebral Microcirculation, Taishan Medical College.MaterialsSixty involved male adult Kunming mice were provided by the Experimental Animal Center of Shandong University. TNF-α primary antibody, kits for enzyme-linked immunosorbent assay(ELISA) and streptavidin-biotin complex immunohistochemical dyeing kit were purchased from Boster Company(Wuhan). MPO kit was purchased from Jiancheng Bioengineering Institute (Nanjing). Cold light source was developed by Hengfa Co., Ltd. (LG-150, Xuzhou).MethodsThis experiment was carried out in the Laboratory of Cerebral Microcirculation of Taishan Medical College between July 2004 and July 2005. The involved 60 Kunming mice were randomized into 3 groups: normal control group (n =6), sham-operation group (n =6) and model group (n =48). Mice in the model group were observed at 30 minutes, 1, 3, 6, 12, 24, 48 and 72 hours after illumination, separately, 6 mice at each time point. In the model group, mice models of local cerebral infarction were developed as follows: The mice were anesthetized to expose left skulls. Taking 2 mm left to sagittal suture and 2 mm posterior to coronal suture as center, a field with diameter of 3 mm for illumination was set. The optical fiber detecting head of cold light source was vertically close to exposed skull. The mice were injected with rose Bengal for 5 minutes, and then cold light source was open for 10 minutes. Illumination was omitted in the sham-operation group. Mice in the control group were not modeled. At postoperative 6 hours, TNF-α expression in infracted-side cortex was detected with immunohistochemical method and ELISA, and MPO activity in infracted-side cortex with chromatometry. MPO activity could reflect the infiltration degree of neutrophils in tissue. Stronger activity indicated severer infiltration. Single-factor analysis of variance was used for comparison among groups, q test for pairwise comparison and correlative analysis for detecting the inter-parameter correlation.Main outcome measuresChanges in TNF-α expression and MPO activity of left cortex of mice in each group.ResultsSixty mice were involved in the final analysis. After cerebral infarction, TNF-α positive cells were neurons and glial cells mainly, distributing in and around the infarct region. TNF-α expression in cortex of mice of sham-operation group was (615.7±16.1) ng/L, and that of model group increased to (792.2±17.8) ng/L at 3 hours after illumination, and reached peak [(921.9±23.9) ng/L] at 6 hours after illumination, and decreased to (848.0±30.6) ng/L at 12 hours after illumination and recovered to the normal level [(625.3±14.3) ng/L] at 72 hours after illumination. MPO activity of sham-operation group was (7.151±0.433) nkat/g, and that of model group increased to (10.469±0.600) nkat/g at 3 hours after illumination, reached the peak [(15.486±0.650) nkat/g] at 12 hours after illumination, decreased to (11.052±0.617) nkat/g at 24 hours after illumination and recovered to the normal level [(7.418±0.617) nkat/g] at 72 hours after illumination. Change of MPO activity lagged behind that of TNF-α, and correlative analysis showed that the both were positively correlated (r =0.953, P < 0.01).ConclusionIn the acute stage of cerebral infarction of mice induced by photochemical method, TNF-α expression in infarcted-side cortex is closely related with infiltration of neutrophils. TNF-α induces inflammatory cells to intrude into ischemic brain tissue, and participates in the inflammatory reaction process at the early stage of cerebral ischemia.