Email updates

Keep up to date with the latest news and content from BMC Neuroscience and BioMed Central.

Open Access Research article

Anti-apoptotic and neuroprotective effects of Tetramethylpyrazine following spinal cord ischemia in rabbits

Li-Hong Fan*, Kun-Zheng Wang, Bin Cheng, Chun-Sheng Wang and Xiao-Qian Dang

Author Affiliations

Department of Orthopedics, Second Affiliated Hospital Xi'an Jiao Tong University, Xiwu Road, Xi'an, shaanxi, 710004, China

For all author emails, please log on.

BMC Neuroscience 2006, 7:48  doi:10.1186/1471-2202-7-48

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2202/7/48


Received:2 March 2006
Accepted:14 June 2006
Published:14 June 2006

© 2006 Fan et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Tetramethylpyrazine (TMP) is one of the most important active ingredients of a Chinese herb Ligusticum wallichii Franchat, which is widely used in many ischemia disorders treatments. However, the exact mechanism by which TMP protects the spinal cord ischemia/reperfusion (I/R) injury is still unknown. For this purpose, rabbits were randomly divided into sham group, control group and TMP group. After the evaluation of neurologic function, the spinal cords were immediately removed for biochemical and histopathological analysis. Apoptosis was measured quantitatively by the terminal transferase UTP nick end-labeling (TUNEL) method and confirmed by electron microscopic examination, the expression of Bax and Bcl-2 was immunohistochemically evaluated and quantified by Western blot analysis.

Results

Neurologic outcomes in the TMP-group were significantly better than those in the control group (P < 0.05). TMP decreased spinal cord malondialdehyde (MDA) levels and ameliorated the down regulation of spinal cord superoxide dismutase (SOD) activity. TMP significantly reduced the loss of motoneurons and TUNEL-positive rate. Greater Bcl-2 and attenuated Bax expression was found in the TMP treating rabbits.

Conclusion

These findings suggest that TMP has protective effects against spinal cord I/R injury by reducing apoptosis through regulating Bcl-2 and Bax expression.

Background

Spinal cord ischemia/reperfusion (I/R) injury may present immediate or delayed paraplegia that occurs 4% to 33% of patients undergoing surgery on the thoracic aorta [1]. Therefore, In attempt to prevent this complication, various methods of spinal cord protection have been suggested, including temporary shunts or partial bypass, hypothermia, drainage of cerebrospinal fluid, and pharmacologic measures [2-4]. Despite their use, paraplegia remains a persistent complication[5].

Although the exact mechanism of I/R injury is not fully understood, it is believed that Oxidative stress plays a pivotal role in triggering lipid peroxidation, DNA damage and specific gene expression [6]. In addition, blood-brain-barrier disruption, mediated by oxygen free radicals, results in spinal cord edema[7]. Oxidative stress resulting from reactive oxygen species (ROS) production is also implicated in apoptosis. Although ischemic neuronal cell death had been traditionally interpreted by necrotic mechanisms, the role of apoptotic mechanisms has been recently proposed in neuronal cell death following spinal cord I/R injury [8]. Several studies have suggested that apoptotic mechanisms were initiated at the molecular level in I/R neural cells[9,10].

In traditional Oriental medicine, Ligusticum wallichii Franchat (Chuan Xiong) is applied in the treatment of neurovascular and cardiovascular diseases. Tetramethylpyrazine (TMP), a purified and chemically identified component of Chuan Xiong, has strong effects to scavenge oxygen free radicals [11]. It has been shown that TMP can alleviate kidney and brain damage induced by I/R via scavenging free radicals[12,13]. However it remains uncertain whether the protective effects of TMP on spinal cord I/R injury are related to scavenging free radicals and suppressing apoptotic pathways.

In this study, the authors investigated the effect of TMP on the neurologic function, biochemical and histopathological changes and studied its impact on expression of pro- and anti-apoptotic proteins as well as the numbers of apoptotic cells following spinal cord I/R injury in rabbits.

Methods

All experimental protocols were approved by our Institutional Committee on Animal Research, and were carried out in accordance with the National Institutes of Health guidelines for animal use and care (National Institutes of Health publication no. 96- 23, revised 1996). Experiments were performed on 36 adult male New Zealand White rabbits (provided by Experimental Animal Center of the Xi'an Jiaotong University) weighing 2.5 to 3.0 kg. The animals were initially anaesthetised with pentobarbital sodium (30 mg/kg IV, sigma, USA, NO: 20030709), followed by a half-dose as required during surgical procedure. No animals received hemodynamic or ventilatory support. The left ear vein was cannulated with a 24-gauge catheter for intravenous drug administration. The right femoral artery was catheterized for blood pressure and heart rate monitoring (Spacelab, USA, model 90206A). Arterial blood was sampled for determination of blood gases (AVL-2, Switzerland) and blood glucose (One Touch II, USA). The rectal body temperature was maintained close to 38°C with the aid of a heating pad during the study.

Experimental groups and Animal models

Rabbits were randomly assigned to 3 groups (n = 12 each). In the TMP group, TMP (30 mg/kg) (Changzhou Pharmacological Co., China, NO: 99091401) was injected via ear vein 30 min before aortic clamping and at the onset of reperfusion. Control animals underwent standard aortic occlusion and intravenous injection of 0.9% sodium chloride under conditions identical to the TMP injection. Sham operated animals subjected to operative dissections without aortic occlusion.

Each group of animals was divided into four experimental subgroups: group A for Biochemical analysis (n = 3), group B for hematoxylin and eosin staining (H&E), Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End-Labeling (TUNEL) staining and immunohistochemistry (n = 3), group C for electron microscopy (n = 2), group D for Western blot assay (n = 4). The rabbit model of spinal cord I/R injury was established according to Savas'discription [14]. Briefly, after sterile preparation, a 10-cm midline incision was performed. Following anticoagulation with 400 unit's heparin, the abdominal aorta was cross-clamped at the level just inferior to the origin of the left renal artery and at the level of aortic bifurcation for 30 min. Reperfusion was initiated by removal of the occlusion and lasted 48 h. The abdomen was then closed.

Neurologic evaluation

Neurological function was observed at the 24th and 48th hour after reperfusion according to Johnson's score[15].

0: Hind-limb paralysis;

1: Severe paraparesis;

2: Functionalmovement, no hop;

3: Ataxia, disconjugate hop;

4: Minimal ataxia;

5: Normal function.

Two individuals without knowledge of the treatment graded neurological function independently.

Histological study

The animals were euthanized by intravenous administration of a high concentration of pentobarbital at the 48th hour and the spinal cords were quickly removed. The spinal cords were immersed in 4% paraformaldehyde in 0.1 mol/l phosphate buffer and stored at 4°C for 2 weeks. The specimens for microscopy were prepared by obtaining spinal cord cross sections from the L2 or L3 vertebra. The specimens were then embedded in paraffin, cut into sections of 5μm thickness, stained with hematoxylin-eosin (H&E). The specimens were examined under the light microscope by a neuropathologist who was blinded to the study.

Preparation for electron microscopic examination of excised cords

The specimens were fixed in 2.5% glutaraldehyde for 6 h, washed in phosphate buffer (pH 7.4), postfixed in 1% osmium tetroxide in phosphate buffer (pH 7.4), and dehydrated in increasing concentrations of alcohol. Then the tissues were immersed in propylene oxide and embedded in epoxy resin embedding media. Ultrathin sections (thickness 60 nm) were cut and stained with uranyl acetate and lead citrate, and examined with a ZEISS-EM902 transmission electron microscope (Carl Zeiss, Thornwood, NY).

Biochemical analysis

Spinal cord tissues were washed two times with cold saline solution and stored in a deep freeze kept at -30°C until analysis. Tissue MDA levels were determined by the method described by Wasowicz[16]. Briefly, MDA was reacted with thiobarbituric acid by incubating for 1 h at 95–100°C. Following the reaction, fluorescence intensity was measured in the n-butanol phase with a fluorescence spectrophotometry(Hitachi, Model F-4010, Japan), by comparing with a standard solution of 1,1,3,3 tetramethoxypropane. Results were expressed in terms of nmol/g wet tissue. Total (Cu-Zn and Mn) SOD activity was measured by reduction of nitrobluetetrazolium (NBT) by xanthine-xanthine oxidase system. Enzyme activity leading to 50% inhibition was accepted as one unit. Results were expressed as U/mg protein [17]. Protein concentrations were determined according to Lowry's method [18].

TUNEL staining and immunohistochemistry

TUNEL staining was performed on paraffin sections using an in situ cell death detection kit (Rochev, Germany) according to the manufacturer's instructions. Sections were counterstained with hematoxylin. A negative control was similarly performed except for omitting TUNEL reaction mixture. Only cells showing nuclear condensation/fragmentation and apoptotic bodies in the absence of cytoplasmic TUNEL reactivity were considered apoptotic. For immunohistochemistry, sections, blocked using 2% normal goat serum in PBS, were incubated for overnight at 4°C with mouse monoclonal antibody against Bcl-2/Bax at a dilution of 1:50 (Maxim Biotech Inc, China) followed by followed by a biotinylated sheep anti-mouse antibody and avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA.) for 2 h. The slices were colorized with DAB/H2O2 solution, and then cell nucleuses were counterstained with hematoxylin. Each procedure was followed by several rinses in PBS. Blank staining was carried out in the same way as the above, except for eliminating the primary antibodies. Brown color of nuclei was taken as the positive staining of apoptotic neuronal cells and Brown color of cytoplasm was taken as the positive staining of Bcl-2/Bax. For quantitative analysis, 10 microscopic fields were taken, and all neurons, including neurons with TUNEL staining were counted. The mean values of the percentage of neurons with TUNEL positive staining were taken for further processing.

Western blot assay of Bcl-2 and Bax proteins

Spinal cord tissue was placed in lysis buffer containing inhibitors(leupeptin, pepstatin A, and aprotinin), homogenized, and then centrifuged(12,000 × g). After determining concentration of protein in each sample using a protein assay (Bio-Rad, Hercules, CA, USA), Samples were loaded (50 mg of protein/lane), electrophoresed on a 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted to a nylon filter. Blots were probed with mouse monoclonal antibody against Bcl-2/Bax at a dilution of 1:200 (Maxim Biotech Inc, China) and visualized with horseradish peroxidase-conjugated secondary antibodies by enhanced chemiluminescence detection reagents (Amersham). Bcl-2 and Bax proteins were detected as 26 and 21 kDa bands, respectively, using molecular weight marker bands. The filter was scanned by FluorImager 595 (Amersham) and quantified with NIH Image J.

Statistical analysis

Statistical analysis was performed using SPSS 10.0. An unpaired t-test was used for comparisons in physiological parameters, MDA levels, SOD activity, TUNEL-positive rate and Bcl-2/Bax expression between the groups. Neurological scores were analyzed with nonparametric method (Kruskal-Wallis test) followed by the Mann-Whitney U test with Bonferroni correction. Data were expressed as mean ± S.D. and statistical significance was set at P <0.05.

Results

Physiological parameters

Physiological variables were within normal limits at any evaluating time points, and showed no statistically significant differences between the groups [see 1].

Additional File 1. Pysiologic Variables MABP: mean arterial blood pressure; RT: rectal temperature; BG: blood glucose level. There were no statistically significant differences in any physiological parameters between the groups.

Format: XLS Size: 17KB Download file

This file can be viewed with: Microsoft Excel ViewerOpen Data

Neurologic function evaluation

The results are shown in Table 1. No neurologic anomaly was observed at the 24th and 48th hours after reperfusion in sham group, except a mild alteration in one animal. The values of Johnson's score in TMP group and control group were significantly lower in comparison with sham group at the 24th hour. The values of control group were significantly lower at the 48th hour in comparison with the same group. Another finding was that, at both the 24th and 48th hour, the values of the TMP group were significantly better in comparison with the control group.

Table 1. Changes in neurologic outcome at the 24 th and 48th hour reperfusion

Histopathologic study

Representative photographs of HE-stained sections are shown in Fig 1. No sign of histopathologic abnormalities was observed in sham-operated rabbits with normal motor function (Figure1A). However, the spinal cords from rabbits in control group that suffered paraplegia (Johnson score 1) exhibited necrotic changes with karyolysis and neurophil vacuolation (Figure1B). The spinal cords of the rabbits rated Johnson score 4 in TMP group showed mild degrees of destruction such as triangular shape, and Nissl granule loss in some motor neurons (Figure1C).

thumbnailFigure 1. Spinal cord histopathology following I/R. Rabbits were treated with vehicle (saline) or TMP (30 mg/kg) prior to the onset of ischemia (30 min), followed by 48 h of reperfusion. Sham animals received the same pretreatment as I/R group, followed by sham operation. The ischemic spinal cord sections were prepared and stained with H&E. Figure A represent the sham rabbits, which show normal histology. Figure B represent control rabbits, which show a pattern of necrotic changes with karyolysis and neurophil vacuolation. Figure C represent TMP- treated rabbits, which show mild degrees of destruction such as triangular shape, and Nissl granule loss in some motor neurons. Figures(magnification × 200) are representative of 3 separate experiments with similar results.

Electron microscopy

Under transmission electron microscopy, nucleus of neuron in sham group displayed normal morphology, including normal shape of nuclei and evenly distributed nuclear chromtin(Figure2A). In control group, neuron showed features of apoptosis, including nucleus shrinkage, dense aggregation of chromatin (Figure2B, arrows) and chromatin margination (Figure2C, arrows),. The results in Figure2B and Figure2C indicate that neural cell apoptosis occurred in the spinal cord at 48 h following 30 min ischemia.

thumbnailFigure 2. Transmission Electron microscopic evidence of neuronal apoptosis in the ventral horn of the spinal cord. The rabbit spinal cords were fixed by transcardial perfusion and removed at 48 h reperfusion, or operation for sham control and processed as described in Experimental Procedures. A, sham control (magnification × 10000); B-C, I/R control (magnification × 20000). I/R induced neuronal apoptosis, as demonstrated by specific morphological features. No apoptotic neurons were found in sham group sections. N, nucleus; nc, nucleolus; C, cytoplasm; M, mitochondria. Figures are representative of 3 separate experiments with similar results.

The biochemical analysis of oxidant stress markers in spinal cord

A significant decrease in SOD activities in the control group was determined when compared to that of sham group (p < 0.01). TMP treatment significantly prevented the decreases in the SOD activities produced by I/R (Figure3). I/R produced a significant increase in MDA level in spinal cord when compared with sham group (p < 0.01). I/R-induced increments in MDA content were significantly prevented by TMP Treatment(Figure 4).

thumbnailFigure 3. Effects of TMP on SOD Activity at the 48th hour reperfusion. The Cu/Zn-SOD activity of spinal cord in sham group, Control group and TMP group was determined as described under "Methods". Average value and SD are shown, N = 3.*P < 0.01, vs sham group; **P < 0.01, vs control group.

thumbnailFigure 4. Effects of TMP on MDA Level at the 48th hour reperfusion. The MDA level of spinal cord in sham group, Control group and TMP group was determined as described under Methods. Average value and SD are shown, N = 3.*P < 0.01, vs sham group; **P < 0.01, vs control group.

TUNEL staining and immunohistochemistry for Bax and Bcl-2

No TUNEL-positive cells were detected in sham group (Figure5A), whereas many cells were intensely stained in the anterior horn of spinal cord after I/R (control group) (Figure5B). However, TMP treatment decreased staining and reduced the number of TUNEL-positive cells (Figure5C). Representative microphotograph of immunohistochemistry staining for Bax and Bcl-2 are shown in Figure 6, 7 respectively. The expression of Bax was weak in the sham group (Figure6A) and more Bax-positive neurons in the control group (Figure6B) than in TMP-treated animals (Figure6C). The expression of Bcl-2 was strong in sham group (Figure7A) and moderate Bcl-2 expression in the control group (Figure7B) compared with the strong up-regulation of Bcl-2 in the TMP-treated group (Figure7C).

thumbnailFigure 5. Effects of TMP on cell apoptosis in the spinal cord at the 48th hour reperfusion. Representative images of TUNEL staining (magnification × 200) in sham group (A), Control group (B) and TMP groups (C). Quantitative analysis of apoptosis rate (D). Cell apoptosis was determined using TUNEL staining as described under "Methods". Cell apoptosis rate is expressed as the mean ± S.D. from three experiments. *P < 0.01, vs sham group; **P < 0.01, vs control group.

thumbnailFigure 6. Effects of TMP on Bax expression in spinal cord at the 48th hour reperfusion. Immunohistochemical photomicrographs (magnification × 400) of anterior horn tissue stained for Bax protein in sham group (A), control group (B) and TMP group (C). Immunostaining was performed using a specific anti- Bax antibody and developed with stable DAB. The positive staining of Bax is presented by a brown color of cytoplasm. Figures are representative of 3 separate experiments with similar results.

thumbnailFigure 7. Effects of TMP on Bcl-2 expression in spinal cord at the 48th hour reperfusion. Immunohistochemical photomicrographs (magnification × 400) of anterior horn tissue stained for Bcl-2 protein in sham group (A), control group (B) and TMP group (C). Immunostaining was performed using a specific anti- Bcl-2 antibody and developed with stable DAB. The positive staining of Bcl-2 is presented by a brown color of cytoplasm. Figures are representative of 3 separate experiments with similar results.

Expression in Bcl-2 and Bax proteins

Expression of Bcl-2 and Bax proteins was visualized by Western blot analysis as shown in Figure 8. Spinal cord ischemia reperfusion obviously reduced Bcl-2 expression and increased Bax expression compared with the sham group. Treatment with TMP was associated with greater Bcl-2 and attenuated Bax expression relative to the vehicle control group.

thumbnailFigure 8. Effect of TMP on expression of Bcl-2/Bax proteins in spinal cord at the 48th hour reperfusion. Western analysis was carried out as described under Methods and the blots are shown in the upper right hand corner. Lane 1 represents sham group; Lane 2 represents vehicle control group; Lane 3 represents TMP group. The bars depict densitometry analyses of Western Blots from four independent experiments. Ischemia reperfusion obviously reduced Bcl-2 expression and increased Bax expression compared with the sham group. *P < 0.01, vs sham group; **P < 0.01, vs control group.

Discussion

Neuroprotective effects of TMP

This study demonstrates a considerable neuropotective effect of TMP, an active ingredient of the Chinese herb Ligusticum wallichii Franchat, on neurological, biochemical and histopathological status of spinal cord I/R in rabbits. There is increasing evidence that free radicals are generated by I/R and they contribute to tissue injury [19]. ROS attack a variety of critical biological molecules, including membrane lipids, essential cellular proteins, and DNA[20]. We studied the effect of TMP on lipid peroxidation, which was measured in terms of MDA. TMP reversed the increase in MDA levels to a considerable extent, thereby confirming its antioxidant role in I/R. Furthermore, we showed that SOD levels increased following TMP treatment. The SOD is the first line of defense against free radical generation. It has been reported that total SOD is down-regulated following spinal cord I/R [21]. Decreased SOD renders a tissue susceptible to oxidant injury. Therefore, the elevated SOD levels induced by TMP may contribute to reduce superoxide radicals following spinal cord I/R.

In our study, the histology of the spinal cords confirms the clinical observations. In general, severity of injury correlated well with the degree of neuronal damage. In animals that had significant impairment of motor function, evidence of both necrosis and apoptosis was apparent. However, TMP increased the proportion of animals that had normal motor function, and in these animals, necrosis was decreased and more normal motoneurons were preserved. This improvement of neurologic function and the histopathological findings reveal the protective effect of TMP on spinal tissue against I/R injury.

Bax/Bcl-2 dependent anti-apoptotic effects of TMP

The principal finding of this work is that TMP increased Bcl-2 expression together with significant decrease in Bax expression in spinal cord. In addition, TMP significantly reduced the number of TUNEL-positive cells in anterior horn of the spinal cord, and the Bax/Bcl-2 expression appeared to correlate with the anti-apoptotic effect.

It has been suggested that neuronal apoptosis occurs concurrently with necrosis following spinal cord I/R and may contribute predominantly to delayed onset of neuronal cell death [22,23]. The major mechanism of I/R induced apoptosis is attributed to the ROS release. ROS induces apoptosis by causing DNA damage, oxidation of lipid membranes, and activation of the proteins responsible for apoptosis[24,25]. Among these apoptosis regulatory proteins, the Bcl-2 family consists of both cell death promoters and cell death preventers. The ratio of anti- to pro-apoptotic molecules such as Bcl-2/Bax determines the response to a death signal. Indeed, the role of the Bcl-2 family in regulating apoptosis has been characterized in CNS ischemia[26,27]. In addition, over-expression of Bcl-2 may play a protective role in neuropathological sequelae after CNS insults [28].

Recent studies have revealed that antioxidants attenuated ischemic neuronal apoptosis through Bcl-2 up-regulation parallel to Bax down-regulation [29]. TMP has been reported to attenuate oxidative damage and apoptosis both in vitro and in vivo [30,31]. In the present study, treatment with TMP is related to an up-regulated level of the anti-apoptotic protein Bcl-2 and a down-regulated pro-apoptotic protein Bax, suggesting that TMP exhibit an inhibitory effect on apoptotic cell death due to spinal cord I/R through modulation of Bcl-2 family.

Conclusion

TMP shows a potent protection against spinal cord I/R injury in rabbit model, and reduces apoptotic cell death through Bcl-2 up-regulation parallel to Bax down-regulation.

Authors' contributions

Li-Hong Fan carried out all in vivo studies, participated in the design of the study and contributed to manuscript preparation. Kun-Zheng Wang assisted in the design of the study, reviewed all data, and assisted in writing the manuscript. Bin Cheng assisted in histopathologic analysis and neurological testing. Chun-Sheng Wang performed all the statistical analysis. Xiao-Qian Dang helped the design of the study and participated in writing the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

The support of Xi'an Jiao Tong University is acknowledged. We thank Prof. Kun-Zheng Wang for the generous supply of Tetramethylpyrazine.

References

  1. Svensson LG, Von Ritter CM, Groeneveld HT, Rickards ES, Hunter SJ, Robinson MF, Hinder RA: Cross-clamping of the thoracic aorta. Influence of aortic shunts, laminectomy, papaverine, calcium channel blocker, allopurinol, and superoxide dismutase on spinal cord blood flow and paraplegia in baboons.

    Ann Surg 1986, 204:38-47. PubMed Abstract | PubMed Central Full Text OpenURL

  2. Svensson LG, Crawford ES, Hess KR, Coselli JS, Safi HJ: Experience with 1509 patients undergoing thoracoabdominal aortic operations.

    J Vasc Surg 1993, 17:357-368. PubMed Abstract | Publisher Full Text OpenURL

  3. Tabayashi K, Niibori K, Konno H, Mohri H: Protection from postischemic spinal cord injury by perfusion cooling of the epidural space.

    Ann Thorac Surg 1993, 56:494-498. PubMed Abstract OpenURL

  4. McCullough JL, Hollier LH, Nugent M: Paraplegia after thoracic aortic occlusion: influence of cerebrospinal fluid drainage. Experimental and early clinical results.

    J Vasc Surg 1988, 7:153-160. PubMed Abstract | Publisher Full Text OpenURL

  5. Zvara DA: Thoracoabdominal aneurysm surgery and the risk of paraplegia: contemporary practice and future directions.

    J Extra Corpor Technol 2002, 34:11-17. PubMed Abstract OpenURL

  6. Chan PH: Role of oxidants in ischemic brain damage.

    Stroke 1996, 27:1124-1129. PubMed Abstract | Publisher Full Text OpenURL

  7. Orendacova J, Marsala M, Marsala J: The blood-brain barrier permeability in graded postischemic spinal cord reoxygenation in rabbits.

    Neurosci Lett 1991, 128:143-146. PubMed Abstract | Publisher Full Text OpenURL

  8. Lin R, Roseborough G, Dong Y, Williams GM, Wei C: DNA damage and repair system in spinal cord ischemia.

    J Vasc Surg 2003, 37:847-858. PubMed Abstract | Publisher Full Text OpenURL

  9. Sakurai M, Nagata T, Abe K, Horinouchi T, Itoyama Y, Tabayashi K: Survival and death-promoting events after transient spinal cord ischemia in rabbits: induction of Akt and caspase3 in motor neurons.

    J Thorac Cardiovasc Surg 2003, 125:370-377. PubMed Abstract | Publisher Full Text OpenURL

  10. Sakurai M, Takahashi G, Abe K, Horinouchi T, Itoyama Y, Tabayashi K: Endoplasmic reticulum stress induced in motor neurons by transient spinal cord ischemia in rabbits.

    J Thorac Cardiovasc Surg 2005, 130:640-645. PubMed Abstract | Publisher Full Text OpenURL

  11. Zhang ZH, Yu SZ, Wang ZT, Zhao BL, Hou JW, Yang FJ, Xin WJ: Scavenging effects of tetramethylpyrazine on active oxygen free radicals.

    Zhongguo Yao Li Xue Bao 1994, 15:229-231. PubMed Abstract OpenURL

  12. Feng L, Xiong Y, Cheng F, Zhang L, Li S, Li Y: Effect of ligustrazine on ischemia-reperfusion injury in murine kidney.

    Transplant Proc 2004, 36:1949-1951. PubMed Abstract | Publisher Full Text OpenURL

  13. Liao SL, Kao TK, Chen WY, Lin YS, Chen SY, Raung SL, Wu CW, Lu HC, Chen CJ: Tetramethylpyrazine reduces ischemic brain injury in rats.

    Neurosci Lett 2004, 372:40-45. PubMed Abstract | Publisher Full Text OpenURL

  14. Savas S, Delibas N, Savas C, Sutcu R, Cindas A: Pentoxifylline reduces biochemical markers of ischemia-reperfusion induced spinal cord injury in rabbits.

    Spinal Cord 2002, 40:224-229. PubMed Abstract | Publisher Full Text OpenURL

  15. Johnson SH, Kraimer JM, Graeber GM: Effects of flunarizine on neurological recovery and spinal cord blood flow in experimental spinal cord ischemia in rabbits.

    Stroke 1993, 24:1547-1553. PubMed Abstract OpenURL

  16. Wasowicz W, Neve J, Peretz A: Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage.

    Clin Chem 1993, 39:2522-2526. PubMed Abstract OpenURL

  17. Sun Y, Oberley LW, Li Y: A simple method for clinical assay of superoxide dismutase.

    Clin Chem 1988, 34:497-500. PubMed Abstract OpenURL

  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent.

    J Biol Chem 1951, 193:265-275. PubMed Abstract | Publisher Full Text OpenURL

  19. Agee JM, Flanagan T, Blackbourne LH, Kron IL, Tribble CG: Reducing postischemic paraplegia using conjugated superoxide dismutase.

    Ann Thorac Surg 1991, 51:911-914. PubMed Abstract OpenURL

  20. Kempski OS: Neuroprotection. Models and basic principles.

    Anaesthesist 1994, (Suppl 2):25-33. OpenURL

  21. Erten SF, Kocak A, Ozdemir I, Aydemir S, Colak A, Reeder BS: Protective effect of melatonin on experimental spinal cord ischemia.

    Spinal Cord 2003, 41:533-538. PubMed Abstract | Publisher Full Text OpenURL

  22. Sakurai M, Hayashi T, Abe K, Sadahiro M, Tabayashi K: Delayed selective motor neuron death and fas antigen induction after spinal cord ischemia in rabbits.

    Brain Res 1998, 797:23-28. PubMed Abstract | Publisher Full Text OpenURL

  23. Hayashi T, Sakurai M, Abe K, Sadahiro M, Tabayashi K, Itoyama Y: Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia.

    Stroke 1998, 29:1007-1012. PubMed Abstract | Publisher Full Text OpenURL

  24. Kroemer G: The proto-oncogene Bcl-2 and its role in regulating apoptosis.

    Nat Med 1997, 3:614-620. PubMed Abstract | Publisher Full Text OpenURL

  25. Galang N, Sasaki H, Maulik N: Apoptotic cell death during ischemia/reperfusion and its attenuation by antioxidant therapy.

    Toxicology 2000, 148:111-118. PubMed Abstract | Publisher Full Text OpenURL

  26. Schabitz WR, Sommer C, Zoder W, Kiessling M, Schwaninger M, Schwab S: Intravenous brain-derived neurotrophic factor reduces infarct size and counterregulates Bax and Bcl-2 expression after temporary focal cerebral ischemia.

    Stroke 2000, 31:2212-2217. PubMed Abstract | Publisher Full Text OpenURL

  27. Wang LM, Yan Y, Zou LJ, Jing NH, Xu ZY: Moderate hypothermia prevents neural cell apoptosis following spinal cord ischemia in rabbits.

    Cell Res 2005, 15:387-393. PubMed Abstract | Publisher Full Text OpenURL

  28. Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK: Bcl-2 overexpression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity.

    J Neurochem 2003, 85:1026-1036. PubMed Abstract OpenURL

  29. Amemiya S, Kamiya T, Nito C, Inaba T, Kato K, Ueda M, Shimazaki K, Katayama Y: Anti-apoptotic and neuroprotective effects of edaravone following transient focal ischemia in rats.

    Eur J Pharmacol 2005, 516:125-130. PubMed Abstract | Publisher Full Text OpenURL

  30. Zhang Z, Wei T, Hou J, Li G, Yu S, Xin W: Iron-induced oxidative damage and apoptosis in cerebellar granule cells: attenuation by tetramethylpyrazine and ferulic acid.

    Eur J Pharmacol 2003, 467:41-47. PubMed Abstract | Publisher Full Text OpenURL

  31. Kao TK, Ou YC, Kuo JS, Chen WY, Liao SL, Wu CW, Chen CJ, Ling NN, Zhang YH, Peng WH: Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats.

    Neurochem Int 2006, 48:166-176. PubMed Abstract | Publisher Full Text OpenURL