Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury Lihong Fan, MD, Kunzheng.

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Tetramethylpyrazine protects spinal cord and reduces inflammation in a rat model of spinal cord ischemia-reperfusion injury Lihong Fan, MD, Kunzheng Wang, MD, Zhibin Shi, MD, Jun Die, MD, Chunsheng Wang, MD, Xiaoqian Dang, MD Journal of Vascular Surgery Volume 54, Issue 1, Pages 192-200 (July 2011) DOI: 10.1016/j.jvs.2010.12.030 Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 1 Neurologic function evaluated using the Basso, Beattie, and Bresnahan (BBB) scores during 48 hours after transient ischemia of the spinal cord. *P < .01 vs sham group at every time point. #P < .05 vs control group at the time point of 6, 12, 24, and 48 hours. Data are presented as mean ± SEM (n = 6 each group). TMP, Tetramethylpyrazine. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 2 Top panel, Triphenyltetrazolium chloride staining of representative spinal cord tissue sections 48 hours after reperfusion: Spinal cord tissue in the lumbar enlargements from the sham group (S), control group (C) and tetramethylpyrazine (TMP) group (T). Bottom panel, Quantitative analysis of spinal cord infarction zone. The infarction size values are expressed as the mean ± SEM. *P < .01 vs sham group; #P < 0.01 vs control group. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 3 Nissl staining of spinal cord tissue sections from rats in sham group (S), control group (C), and tetramethylpyrazine (TMP) group (T) (original magnification ×400; n = 6 per group). The well-maintained nerve cells in the gray matter of spinal cord were seen in sham group, whereas the control group showed a large population of swollen nerve cells with vacuolated cytoplasm and disintegrated nucleus. Treatment with TMP markedly reduced these pathologic damages. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 4 Myeloperoxidase (MPO) activities in spinal cord tissues of the three groups (n = 6 per group). Spinal cord tissue homogenates were obtained from the ischemic section, and the MPO activity was measured 48 hours after reperfusion. The data are expressed as mean ± SEM. *P < .01 compared with sham group, #P < .01 compared with control group. TMP, Tetramethylpyrazine. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 5 Immunohistochemical localizations of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-10 in spinal cord 48 hours after reperfusion in sham group (S), control group (C), and tetramethylpyrazine (TMP) group (T) (original magnification ×200; n = 6 rats per group). No positive staining of IL-1β, TNF-α and IL-10 was observed in spinal cord tissues from sham group; a substantial increase in the release of IL-1β, TNF-α, and IL-10 was found in various cells in the ischemic spinal cord tissues from the control group; and IL-1β and TNF-α decreased significantly, whereas IL-10 production increased remarkably in the TMP group compared with the control group. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 6 The contents of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-10 in the spinal cords of the three groups. The contents were measured by enzyme-linked immunosorbent assay. The expression levels of IL-1β, TNF-α, and IL-10 in spinal cord tissue increased significantly in the control group compared with those in the sham group; the proinflammatory cytokines IL-1β and TNF-α in the tetramethylpyrazine (TMP) group were significantly decreased compared with those in control group; IL-10 production was significantly increased by treatment with TMP. Data are expressed as mean ± SEM, n = 6 per group. *P < .01 compared with sham group and #P < .01 compared with control group. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 7 Immunohistochemistry staining of nuclear factor (NF)-κB (p65) in spinal cord 48 hours after reperfusion in sham group (S), control group (C), and tetramethylpyrazine (TMP) group (T; original magnification ×400). Positive cells were defined as hanging buffy grains in the cellular nucleus and cytoplasm (n = 6 rats per group). NF-κB expression could be observed periodically in sham group (S); NF-κB (p65) was predominantly located in the nucleus in control group (C); TMP could significantly inhibit its translocation from cytoplasm into the nucleus (T). Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 8 The nuclear factor (NF)-κB (p65) protein levels in the spinal cord tissues of the three groups 48 hours after reperfusion. The NF-κB (p65) protein levels were determined with specific antibody by Western blot. Densitometric analysis was normalized to β-actin. Results were representative of three independent experiments. The bar graph demonstrates that the NF-κB expression was upregulated after spinal cord ischemia-reperfusion injury, and that treatment with tetramethylpyrazine (TMP) significantly decreased the NF-κB expression. *P < .01 compared with sham group and #P < .01 compared with control group. Journal of Vascular Surgery 2011 54, 192-200DOI: (10.1016/j.jvs.2010.12.030) Copyright © 2011 Society for Vascular Surgery Terms and Conditions