- Email: [email protected]
reperfusion injury in rabbits
Journal of Clinical Neuroscience 17 (2010) 486–489
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory Study
Neuroprotective effect of mesna (2-mercaptoethane sulfonate) against spinal cord ischemia/reperfusion injury in rabbits Habibullah Dolgun a, Zeki Sekerci a, Erhan Turkoglu a,*, Hayri Kertmen a, Erdal R. Yilmaz a, Murat Anlar b, Imge B. Erguder c, Hakan Tuna d a
First Neurosurgery Clinic, Diskapi Yildirim Beyazit Education and Research Hospital, Ankara, Turkey Second Pathology Clinic, Diskapi Yildirim Beyazit Education and Research Hospital, Ankara, Turkey Department of Biochemistry, Ankara University Faculty of Medicine, Ankara, Turkey d Department of Neurosurgery, Ankara University Faculty of Medicine, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 12 April 2009 Accepted 7 July 2009
Keywords: Apoptosis Caspase Ischemia/reperfusion injury Mesna 2-Mercaptoethane sulfonate
a b s t r a c t Although the precise mechanism by which ischemia/reperfusion injury occurs in the spinal cord remains unclear, it is evident that free oxygen radicals and apoptosis play major roles in the destruction of membrane lipids, damage to DNA and cell death. The apoptotic process involves activation of the caspase-3 cascade. Although it is widely used as a protective agent against cell injury, it is unknown whether mesna (2-mercaptoethane sulfonate) ameliorates neuronal ischemic injury. The aim of this study was to determine the effect of mesna on caspase-3 activity in a rabbit model. Adult rabbits underwent spinal cord ischemic injury via occlusion of the abdominal aorta for 20 min. Twenty-four hours after ischemia, spinal cord samples were obtained and tissue caspase-3 activity was measured. Rabbits that had been given a single dose of 150 mg/kg mesna had decreased caspase-3 activity in the spinal cord following ischemia/ reperfusion injury, indicating a protective effect. However, caspase-3 activity was lower in rabbits given methylprednisolone than in those given mesna, indicating that methylprednisolone has the stronger protective effect of the two agents. Published by Elsevier Ltd.
1. Introduction After injury, oxygen free radicals play a major role in the destruction of membrane lipids, essential cell proteins and DNA, although the exact biomechanism by which ischemia and reperfusion (I/R) injury occurs in spinal cord is not completely understood. Necrosis is considered to be the principal mechanism of cell death after spinal cord injury (SCI).1 This is followed by a progressive injury process (secondary injury) that initiates apoptosis.2 Recent therapeutic interventions have been focused on secondary injury, because definitive neurologic recovery is dependent on the amount of spared functional tissue and its location in the spinal cord.3 Apoptosis is active and genetically programmed cell death, and has been shown to be associated with neurodegenerative disorders, neurotoxicity and neurotrauma.4 The process is primarily regulated by the caspase family of cysteine proteases.5 The apoptotic cell death process involves activation of caspase-3, an intracellular cysteine protease that exists as a proenzyme, becoming activated during the sequence of events associated with apoptosis. In previ-
* Corresponding author. Postal address: Guclukaya Mah, Icozderesi Sok. No. 10/ 20, Ankara 06612, Turkey. Tel.: +90 050 56260200; fax: +90 312 4356745. E-mail address: [email protected] (E. Turkoglu). 0967-5868/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.jocn.2009.07.108
ous studies, a variety of pharmacological agents have been used to prevent apoptosis after experimental injury.3 2-mercaptoethane sulfonate (mesna) is a small, synthetic, highly water-soluble molecule that has the potential to scavenge reactive oxygen species by virtue of its sulfhydryl group.6 It is principally used to reduce the hemorrhagic cystitis induced by cyclophosphamide and ifosfamide, but it is also widely used as a protective agent against chemotherapy toxicity. To the best of our knowledge, there have been no previous investigations of whether mesna ameliorates neuronal injury and neurotoxicity. The main aim of the current study was to evaluate the effect of mesna on caspase-3 activity and to compare its effectiveness with methylprednisolone (MPSS) after experimental spinal cord I/R injury.
2. Materials and methods 2.1. Experimental groups Adult female New Zealand White rabbits weighing 2000–3750 g were used in this study. All experimental procedures used were approved by the ethical committee of Diskapi Yildirim Beyazit Education and Research Hospital. The rabbits were randomized and
H. Dolgun et al. / Journal of Clinical Neuroscience 17 (2010) 486–489
487
blindly assigned to five groups, with seven rabbits per group. The groups were as follows:
1 pmol of substrate per min at 30 °C. The results were expressed as U/mg protein.
1. Control: Rabbits underwent laminectomy and non-ischemic spinal cord samples were obtained immediately after surgery. 2. Trauma: Rabbits underwent transient global spinal cord ischemia. After laminectomy, spinal cord samples were removed at 24 hours post-ischemia. 3. MPSS: As for group 2, but rabbits received a single intravenous dose of 30 mg/kg MPSS (Mustafa Nevzat, Istanbul, Turkey) immediately following spinal cord injury. 4. Mesna: As for group 2, but rabbits received a single intravenous dose of 150 mg/kg mesna (Eczacıbasßı Baxter, Istanbul, Turkey) immediately following spinal cord injury. 5. Vehicle: As for group 2, but rabbits received 1 mL of vehicle solution (saline) intravenously immediately following spinal cord injury.
2.4. Light microscopy
2.2. Surgical procedure and sample preparation The surgical procedure was performed under general anesthesia induced by intramuscular xylazine (10 mg/kg; Bayer, Istanbul, Turkey) and ketamine hydrochloride (60 mg/kg; Parke-Davis, Istanbul, Turkey). After a midline laparotomy, a Yasßargil microvascular clamp (standard aneurysm clamp, FE751; Aesculap, Tuttlingen, Germany) was placed on the aorta 1.0 cm caudal to the left renal artery. Body temperature was monitored and maintained at 37 °C with a heating pad during the operation. I/R injury was applied via occlusion of the abdominal aorta for 20 min, then removal of the clamp. After the surgical and ischemic interventions, the surgical wound was closed in layers with silk sutures. The animals were then allowed free access to water and food at ambient temperature. Twenty-four hours after spinal cord injury, the rabbits were killed using a ketamine overdose. For all animals, L1–L5 laminectomy was performed, with the dura left intact. Spinal cord samples (2 cm) were obtained and maintained at 196 °C (in liquid nitrogen) until colorimetric evaluation. 2.3. Caspase-3 activity Tissues were homogenized in physiological saline (1 g in 5 mL) and centrifuged at 4000g for 20 min. The upper layer of clear supernatant was removed and used in the analyses. Before analysis, the supernatant samples were adjusted so that they contained equal protein concentrations. The protein concentrations of the supernatant samples were measured using the Lowry method.7 The Lowry method depends on the reactivity of the nitrogen in peptides with copper ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteau phosphomolybdic-phosphotungstic acid to heteropolymolybdenum blue by the coppercatalyzed oxidation of aromatic amino acids. Absorbance measurements were made at 700 nm using a spectrophotometer. The protein concentration of the sample was determined using a protein calibrator. The caspase-3 activity of the tissue samples was measured using the Caspase-3 Colorimetric Detection Kit (907-013; Assay Designs, Ann Arbor, MI, USA). The kit involves the conversion of a specific chromogenic substrate for caspase-3 (acetyl-Asp-GluVal-Asp-p-nitroanilide), followed by colorimetric detection of the product (p-nitroaniline) at 405 nm. The absolute value for caspase-3 activity can be determined by comparison with a signal given by the p-nitroaniline calibrator. Activity measurements were quantified by comparing the optical densities obtained with standards with the p-nitroaniline calibrator. One unit of caspase-3 activity was defined as the amount of enzyme needed to convert
The cord specimens obtained at 24 hours post-injury were prepared for histological study. Each cord segment (approximately 1 cm, centered at the injury site) was immersed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer and stored at 4 °C. The specimens were then embedded in paraffin, cut into sections of 5 lm thickness, and stained with hematoxylin-eosin (H&E). The specimens were examined under a light microscope by a neuropathologist who was blinded to the study design. 2.5. Statistical analysis All data collected were coded, recorded and analyzed using SPSS 10.0.1 for Windows (SPSS Inc., Chicago, IL, USA). All data are presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) for parametric data was used for comparing differences between two or more groups. Tukey’s test was used to determine differences between groups. Differences were considered to be significant at p < 0.05. 3. Results There was a statistically significant difference between the Control and Trauma groups with regard to mean caspase-3 activity (p < 0.01). I/R injury clearly elevated caspase-3 activity in the damaged tissue. MPSS prevented an increase in caspase-3 activity and effectively inhibited apoptotic cell death. Mesna treatment also decreased caspase-3 activity relative to the control, but was not as effective as MPSS. There was a statistically significant difference between the MPSS and Mesna groups (p < 0.01). The difference between the Vehicle group and the Trauma group was not statistically significant (p > 0.01). The difference between Vehicle and Mesna groups were not statistically significant (p > 0.01) (Fig. 1). As expected, light microscopic examination of the spinal cord samples from the Control group was normal (Supplementary Fig. 1A). In the Trauma group, diffuse hemorrhage and congestion in the gray matter were observed at 24 h. There was marked hemorrhagic necrosis and widespread edema in both the white and gray matter. In the damaged portion there were infiltrating polymorphonuclear leukocytes, lymphocytes and plasma cells, as well as neuronal pyknosis, a loss of cytoplasmic features, and cytoplasmic eosinophilia (Supplementary Fig. 1B). In the Mesna group, the cord tissue had similar features to that from the Trauma group, although the edema and infiltration with polymorphonuclear leukocytes were relatively mild in both the white and gray matter zones (Supplementary Fig. 1C). 4. Discussion Necrosis and apoptosis are the two major pathways of neuronal death resulting from I/R injury.8,9 Acute ischemia usually leads to necrosis because of a reduction in spinal blood flow accompanied by depletion of adenosine triphosphate reserves and the development of edema.1,9,10 Motor neurons in the spinal cord are selectively more vulnerable to ischemia; however, the exact biomechanism of their selective vulnerability has not been clearly established.10 Similarly, motor neurons in the cerebral cortex, CA1 pyramidal cells in the hippocampus, and Purkinje cells in the cerebellum are also known to be selectively vulnerable to ischemia.11,12 Although early reperfusion can prevent the generalization of necrosis, it may also have several potentially deleterious
488
H. Dolgun et al. / Journal of Clinical Neuroscience 17 (2010) 486–489
Fig. 1. Concentration of caspase-3 in rabbit spinal cord tissue. Data are mean values ± 95% confidence interval. Mesna = 2-mercaptoethane sulfonate, MPPS = methylprednisolone, I/R = ischemia and reperfusion.
effects that are collectively described as reperfusion injury. During the reperfusion period, the remaining motor neurons may recover energy metabolism but nevertheless go on to die between hours and several days later, undergoing delayed cell death.13,14 Mild or severe I/R injury has been shown to trigger apoptosis, which leads to later cell death.2,15,16 Mackey et al. demonstrated that approximately 30–40% of cells show features of apoptosis by 24 hours after ischemia.17 It is possible that some cells begin to die via apoptosis but subsequently succumb to necrosis as the environment becomes more toxic.11 Apoptosis is activated by the cysteine protease family known as the caspases.18 Caspase-3 is an interleukin-converting enzyme, and has been suggested to be the principal effector in the mammalian apoptotic and inflammation pathways.19 Sakuri et al. demonstrated an increase in caspase-3 immunoreactivity in the motor neurons of the spinal cord after 15 min of ischemia.9 As a result of ischemic events, the peak increase in caspase-3 induction triggers DNA fragmentation.10 DNA fragmentation and apoptotic cells have also been identified in the ischemic hemisphere after focal and forebrain ischemia.20 In the present study, we demonstrated that I/R injury increased tissue caspase-3 activity in rabbit spinal cord, which is consistent with previous observations.9,10,17 Given the early colorimetric detection of caspases at 24 hours after injury, we concluded that apoptotic cell death was occurring in these neurons at that point. Our results suggest that delayed neural death associated with spinal cord ischemia may be due, in part, to apoptosis. Thus, anti-apoptotic strategies may be useful in the treatment of acute ischemia, and prevention of apoptosis may result in neurologic recovery. After spinal cord ischemia, the remaining cells may recover metabolic activity upon reperfusion but may die 24–48 hours later.13,16 In any event, putative protective pharmacological agents may inhibit caspase-associated apoptosis and prevent neuronal tissue damage after I/R injury.4,21 Mesna is a well-known systemic protective agent against chemotherapy toxicity.6 Mesna ameliorates hemorrhagic cystitis, intestinal mucosal damage, hepatic cell dysfunction and human proximal tubule cell damage.4,6,22 Ypsilantis et al. found that mesna’s mucoprotective effect is mainly based on its anti-apoptotic activity.22 However, to our knowledge there are no previous data on whether mesna also ameliorates neuronal ischemic injury and neurotoxicity. Depending on the dose, it is possible that mesna could prevent or ameliorate the apoptotic effect of ischemic injury. One of the possible mechanisms of mesna’s anti-apoptotic effect after I/R injury may be its antioxidant effect, as it can act as a scav-
enger of oxygen free radicals.6 Glutamate is a key molecule in cellular metabolism, and it serves as metabolic fuel for other functional pathways in the body. Glutamate deprivation leads to oxidant-induced apoptosis via selective activation of caspases and the activation of N-methyl-D-aspartate (NMDA) receptors.22 Excessive accumulation of glutamate, with subsequent overactivation of NMDA receptors, results in an increase in free calcium levels in the cytoplasm. An influx of calcium increases nitric oxide production and cell membrane lipid peroxidation, resulting in the overproduction of peroxynitrite.23 Peroxynitrite causes DNA damage and activates caspase-3.24 Mesna contains a sulfhydryl (SH) group and as such could prevent cell death induced by caspase activation.6 In addition to caspases, protein kinase C (PKC) and second mitochondria-derived activator of caspases (SMACs) are activated in apoptotic cells.6 PKC can play a role in apoptotic cell death. Thus, it is possible that mesna inhibits other apoptosisinducing pathways upstream of caspase-3 activation. A sufficient supply of extra- or intracellular SH groups may protect the neuron from destruction due to ischemic injury. In conclusion, we have shown that a single 150 mg/kg dose of mesna decreases caspase-3 activity in rabbit spinal cord subjected to I/R injury. However, mesna does not appear to be as neuroprotective as MPSS. Further studies will be necessary to determine whether mesna could be used to provide neuroprotection from apoptotic cell death after I/R injury in humans, and to determine what the optimal dose would be. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jocn.2009.07.108. References 1. Akdemir O, Berksoy I, Karaog˘lan A, et al. Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury. J Clin Neurosci 2008;15:672–8. 2. Okutan O, Solaroglu I, Beskonakli E, et al. Recombinant human erythropoietin decreases myeloperoxidase and caspase-3 activity and improves early functional results after spinal cord injury in rats. J Clin Neurosci 2007;14:364–8. 3. Solaroglu I, Kaptanoglu E, Okutan O, et al. Magnesium sulfate treatment decreases caspase-3 activity after experimental spinal cord injury in rats. Surg Neurol 2005;64:17–21. 4. Barut Sß, Ünlü AY, Karaog˘lan A, et al. The neuroprotective effects of z-DEVD.fmk, a caspase-3 inhibitor, on traumatic spinal cord injury in rats. Surg Neurol 2005;64:213–20. 5. Sener G, Sehirli O, Ercan F, et al. Protective effect of MESNA (2-mercaptoethane sulfonate) against hepatic ischemia/reperfusion injury in rats. Surg Today 2005;35:575–80. 6. Schwerdt G, Kirchhoff A, Freudinger R, et al. Mesna or cysteine prevents chloroacetaldehyde-induced cell death of human proximal tubule cells. Pediatr Nephrol 2007;22:798–803. 7. Lowry O, Rosenbrough N, Farr L, et al. Protein measurement with Folin phenol reagent. J Biol Chem 1951;182:265–75. 8. Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal death. A mitochondrial hypothesis. Stroke 1995;26:1478–89. 9. Sakurai M, Nagata T, Abe K, et al. 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;125:370–7. 10. Hayashi T, Sakuria M, Abe K, et al. Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia. Stroke 1998;29:1007–13. 11. Knoblach SM, Nikolaeva M, Huang X, et al. Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome. J Neurotrauma 2002;19:1155–70. 12. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 1982;11:491–8. 13. Citron BA, Arnold PM, Haynes NG, et al. Neuroprotective effects of caspase-3 inhibition on functional recovery and tissue sparing after acute spinal cord injury. Spine 2008;33:2269–77. 14. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 1982;239:57–69. 15. Hearse DJ, Boli R. Reperfusion induced injury: manifestations, mechanisms, and clinical relevance. Cardiovasc Res 1992;26:101–8. 16. Li M, Ona VO, Chen M, et al. Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience 2000;99:333–42.
H. Dolgun et al. / Journal of Clinical Neuroscience 17 (2010) 486–489 17. Mackey ME, Wu Y, Hu R, et al. Cell death suggestive of apoptosis after spinal cord ischemia in rabbits. Stroke 1997;28:2012–7. 18. Emery E, Aldana P, Bunge MB, et al. Apoptosis after traumatic human spinal cord injury. J Neurosurg 1998;89:911–20. 19. Keane RW, Kraydieh S, Lotocki G, et al. Apoptotic and anti-apoptotic mechanisms following spinal cord injury. J Neuropathol Exp Neurol 2001;60:422–9. 20. Li Y, Chopp M, Jiang N, et al. Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1995;15:389–97.
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21. Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 1991;75:15–26. 22. Ypsilantis P, Tentes I, Assimakopoulos SF, et al. Mesna ameliorates intestinal mucosa damage after ifosfamide administration in the rabbit at a dose-related manner. J Surg Res 2004;121:84–91. 23. Beckman JS. The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 1991;15:53–9. 24. Bao F, Liu D. Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3. Neuroscience 2003;116:59–70.
Contents lists available at ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Laboratory Study
Neuroprotective effect of mesna (2-mercaptoethane sulfonate) against spinal cord ischemia/reperfusion injury in rabbits Habibullah Dolgun a, Zeki Sekerci a, Erhan Turkoglu a,*, Hayri Kertmen a, Erdal R. Yilmaz a, Murat Anlar b, Imge B. Erguder c, Hakan Tuna d a
First Neurosurgery Clinic, Diskapi Yildirim Beyazit Education and Research Hospital, Ankara, Turkey Second Pathology Clinic, Diskapi Yildirim Beyazit Education and Research Hospital, Ankara, Turkey Department of Biochemistry, Ankara University Faculty of Medicine, Ankara, Turkey d Department of Neurosurgery, Ankara University Faculty of Medicine, Ankara, Turkey b c
a r t i c l e
i n f o
Article history: Received 12 April 2009 Accepted 7 July 2009
Keywords: Apoptosis Caspase Ischemia/reperfusion injury Mesna 2-Mercaptoethane sulfonate
a b s t r a c t Although the precise mechanism by which ischemia/reperfusion injury occurs in the spinal cord remains unclear, it is evident that free oxygen radicals and apoptosis play major roles in the destruction of membrane lipids, damage to DNA and cell death. The apoptotic process involves activation of the caspase-3 cascade. Although it is widely used as a protective agent against cell injury, it is unknown whether mesna (2-mercaptoethane sulfonate) ameliorates neuronal ischemic injury. The aim of this study was to determine the effect of mesna on caspase-3 activity in a rabbit model. Adult rabbits underwent spinal cord ischemic injury via occlusion of the abdominal aorta for 20 min. Twenty-four hours after ischemia, spinal cord samples were obtained and tissue caspase-3 activity was measured. Rabbits that had been given a single dose of 150 mg/kg mesna had decreased caspase-3 activity in the spinal cord following ischemia/ reperfusion injury, indicating a protective effect. However, caspase-3 activity was lower in rabbits given methylprednisolone than in those given mesna, indicating that methylprednisolone has the stronger protective effect of the two agents. Published by Elsevier Ltd.
1. Introduction After injury, oxygen free radicals play a major role in the destruction of membrane lipids, essential cell proteins and DNA, although the exact biomechanism by which ischemia and reperfusion (I/R) injury occurs in spinal cord is not completely understood. Necrosis is considered to be the principal mechanism of cell death after spinal cord injury (SCI).1 This is followed by a progressive injury process (secondary injury) that initiates apoptosis.2 Recent therapeutic interventions have been focused on secondary injury, because definitive neurologic recovery is dependent on the amount of spared functional tissue and its location in the spinal cord.3 Apoptosis is active and genetically programmed cell death, and has been shown to be associated with neurodegenerative disorders, neurotoxicity and neurotrauma.4 The process is primarily regulated by the caspase family of cysteine proteases.5 The apoptotic cell death process involves activation of caspase-3, an intracellular cysteine protease that exists as a proenzyme, becoming activated during the sequence of events associated with apoptosis. In previ-
* Corresponding author. Postal address: Guclukaya Mah, Icozderesi Sok. No. 10/ 20, Ankara 06612, Turkey. Tel.: +90 050 56260200; fax: +90 312 4356745. E-mail address: [email protected] (E. Turkoglu). 0967-5868/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.jocn.2009.07.108
ous studies, a variety of pharmacological agents have been used to prevent apoptosis after experimental injury.3 2-mercaptoethane sulfonate (mesna) is a small, synthetic, highly water-soluble molecule that has the potential to scavenge reactive oxygen species by virtue of its sulfhydryl group.6 It is principally used to reduce the hemorrhagic cystitis induced by cyclophosphamide and ifosfamide, but it is also widely used as a protective agent against chemotherapy toxicity. To the best of our knowledge, there have been no previous investigations of whether mesna ameliorates neuronal injury and neurotoxicity. The main aim of the current study was to evaluate the effect of mesna on caspase-3 activity and to compare its effectiveness with methylprednisolone (MPSS) after experimental spinal cord I/R injury.
2. Materials and methods 2.1. Experimental groups Adult female New Zealand White rabbits weighing 2000–3750 g were used in this study. All experimental procedures used were approved by the ethical committee of Diskapi Yildirim Beyazit Education and Research Hospital. The rabbits were randomized and
H. Dolgun et al. / Journal of Clinical Neuroscience 17 (2010) 486–489
487
blindly assigned to five groups, with seven rabbits per group. The groups were as follows:
1 pmol of substrate per min at 30 °C. The results were expressed as U/mg protein.
1. Control: Rabbits underwent laminectomy and non-ischemic spinal cord samples were obtained immediately after surgery. 2. Trauma: Rabbits underwent transient global spinal cord ischemia. After laminectomy, spinal cord samples were removed at 24 hours post-ischemia. 3. MPSS: As for group 2, but rabbits received a single intravenous dose of 30 mg/kg MPSS (Mustafa Nevzat, Istanbul, Turkey) immediately following spinal cord injury. 4. Mesna: As for group 2, but rabbits received a single intravenous dose of 150 mg/kg mesna (Eczacıbasßı Baxter, Istanbul, Turkey) immediately following spinal cord injury. 5. Vehicle: As for group 2, but rabbits received 1 mL of vehicle solution (saline) intravenously immediately following spinal cord injury.
2.4. Light microscopy
2.2. Surgical procedure and sample preparation The surgical procedure was performed under general anesthesia induced by intramuscular xylazine (10 mg/kg; Bayer, Istanbul, Turkey) and ketamine hydrochloride (60 mg/kg; Parke-Davis, Istanbul, Turkey). After a midline laparotomy, a Yasßargil microvascular clamp (standard aneurysm clamp, FE751; Aesculap, Tuttlingen, Germany) was placed on the aorta 1.0 cm caudal to the left renal artery. Body temperature was monitored and maintained at 37 °C with a heating pad during the operation. I/R injury was applied via occlusion of the abdominal aorta for 20 min, then removal of the clamp. After the surgical and ischemic interventions, the surgical wound was closed in layers with silk sutures. The animals were then allowed free access to water and food at ambient temperature. Twenty-four hours after spinal cord injury, the rabbits were killed using a ketamine overdose. For all animals, L1–L5 laminectomy was performed, with the dura left intact. Spinal cord samples (2 cm) were obtained and maintained at 196 °C (in liquid nitrogen) until colorimetric evaluation. 2.3. Caspase-3 activity Tissues were homogenized in physiological saline (1 g in 5 mL) and centrifuged at 4000g for 20 min. The upper layer of clear supernatant was removed and used in the analyses. Before analysis, the supernatant samples were adjusted so that they contained equal protein concentrations. The protein concentrations of the supernatant samples were measured using the Lowry method.7 The Lowry method depends on the reactivity of the nitrogen in peptides with copper ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteau phosphomolybdic-phosphotungstic acid to heteropolymolybdenum blue by the coppercatalyzed oxidation of aromatic amino acids. Absorbance measurements were made at 700 nm using a spectrophotometer. The protein concentration of the sample was determined using a protein calibrator. The caspase-3 activity of the tissue samples was measured using the Caspase-3 Colorimetric Detection Kit (907-013; Assay Designs, Ann Arbor, MI, USA). The kit involves the conversion of a specific chromogenic substrate for caspase-3 (acetyl-Asp-GluVal-Asp-p-nitroanilide), followed by colorimetric detection of the product (p-nitroaniline) at 405 nm. The absolute value for caspase-3 activity can be determined by comparison with a signal given by the p-nitroaniline calibrator. Activity measurements were quantified by comparing the optical densities obtained with standards with the p-nitroaniline calibrator. One unit of caspase-3 activity was defined as the amount of enzyme needed to convert
The cord specimens obtained at 24 hours post-injury were prepared for histological study. Each cord segment (approximately 1 cm, centered at the injury site) was immersed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer and stored at 4 °C. The specimens were then embedded in paraffin, cut into sections of 5 lm thickness, and stained with hematoxylin-eosin (H&E). The specimens were examined under a light microscope by a neuropathologist who was blinded to the study design. 2.5. Statistical analysis All data collected were coded, recorded and analyzed using SPSS 10.0.1 for Windows (SPSS Inc., Chicago, IL, USA). All data are presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) for parametric data was used for comparing differences between two or more groups. Tukey’s test was used to determine differences between groups. Differences were considered to be significant at p < 0.05. 3. Results There was a statistically significant difference between the Control and Trauma groups with regard to mean caspase-3 activity (p < 0.01). I/R injury clearly elevated caspase-3 activity in the damaged tissue. MPSS prevented an increase in caspase-3 activity and effectively inhibited apoptotic cell death. Mesna treatment also decreased caspase-3 activity relative to the control, but was not as effective as MPSS. There was a statistically significant difference between the MPSS and Mesna groups (p < 0.01). The difference between the Vehicle group and the Trauma group was not statistically significant (p > 0.01). The difference between Vehicle and Mesna groups were not statistically significant (p > 0.01) (Fig. 1). As expected, light microscopic examination of the spinal cord samples from the Control group was normal (Supplementary Fig. 1A). In the Trauma group, diffuse hemorrhage and congestion in the gray matter were observed at 24 h. There was marked hemorrhagic necrosis and widespread edema in both the white and gray matter. In the damaged portion there were infiltrating polymorphonuclear leukocytes, lymphocytes and plasma cells, as well as neuronal pyknosis, a loss of cytoplasmic features, and cytoplasmic eosinophilia (Supplementary Fig. 1B). In the Mesna group, the cord tissue had similar features to that from the Trauma group, although the edema and infiltration with polymorphonuclear leukocytes were relatively mild in both the white and gray matter zones (Supplementary Fig. 1C). 4. Discussion Necrosis and apoptosis are the two major pathways of neuronal death resulting from I/R injury.8,9 Acute ischemia usually leads to necrosis because of a reduction in spinal blood flow accompanied by depletion of adenosine triphosphate reserves and the development of edema.1,9,10 Motor neurons in the spinal cord are selectively more vulnerable to ischemia; however, the exact biomechanism of their selective vulnerability has not been clearly established.10 Similarly, motor neurons in the cerebral cortex, CA1 pyramidal cells in the hippocampus, and Purkinje cells in the cerebellum are also known to be selectively vulnerable to ischemia.11,12 Although early reperfusion can prevent the generalization of necrosis, it may also have several potentially deleterious
488
H. Dolgun et al. / Journal of Clinical Neuroscience 17 (2010) 486–489
Fig. 1. Concentration of caspase-3 in rabbit spinal cord tissue. Data are mean values ± 95% confidence interval. Mesna = 2-mercaptoethane sulfonate, MPPS = methylprednisolone, I/R = ischemia and reperfusion.
effects that are collectively described as reperfusion injury. During the reperfusion period, the remaining motor neurons may recover energy metabolism but nevertheless go on to die between hours and several days later, undergoing delayed cell death.13,14 Mild or severe I/R injury has been shown to trigger apoptosis, which leads to later cell death.2,15,16 Mackey et al. demonstrated that approximately 30–40% of cells show features of apoptosis by 24 hours after ischemia.17 It is possible that some cells begin to die via apoptosis but subsequently succumb to necrosis as the environment becomes more toxic.11 Apoptosis is activated by the cysteine protease family known as the caspases.18 Caspase-3 is an interleukin-converting enzyme, and has been suggested to be the principal effector in the mammalian apoptotic and inflammation pathways.19 Sakuri et al. demonstrated an increase in caspase-3 immunoreactivity in the motor neurons of the spinal cord after 15 min of ischemia.9 As a result of ischemic events, the peak increase in caspase-3 induction triggers DNA fragmentation.10 DNA fragmentation and apoptotic cells have also been identified in the ischemic hemisphere after focal and forebrain ischemia.20 In the present study, we demonstrated that I/R injury increased tissue caspase-3 activity in rabbit spinal cord, which is consistent with previous observations.9,10,17 Given the early colorimetric detection of caspases at 24 hours after injury, we concluded that apoptotic cell death was occurring in these neurons at that point. Our results suggest that delayed neural death associated with spinal cord ischemia may be due, in part, to apoptosis. Thus, anti-apoptotic strategies may be useful in the treatment of acute ischemia, and prevention of apoptosis may result in neurologic recovery. After spinal cord ischemia, the remaining cells may recover metabolic activity upon reperfusion but may die 24–48 hours later.13,16 In any event, putative protective pharmacological agents may inhibit caspase-associated apoptosis and prevent neuronal tissue damage after I/R injury.4,21 Mesna is a well-known systemic protective agent against chemotherapy toxicity.6 Mesna ameliorates hemorrhagic cystitis, intestinal mucosal damage, hepatic cell dysfunction and human proximal tubule cell damage.4,6,22 Ypsilantis et al. found that mesna’s mucoprotective effect is mainly based on its anti-apoptotic activity.22 However, to our knowledge there are no previous data on whether mesna also ameliorates neuronal ischemic injury and neurotoxicity. Depending on the dose, it is possible that mesna could prevent or ameliorate the apoptotic effect of ischemic injury. One of the possible mechanisms of mesna’s anti-apoptotic effect after I/R injury may be its antioxidant effect, as it can act as a scav-
enger of oxygen free radicals.6 Glutamate is a key molecule in cellular metabolism, and it serves as metabolic fuel for other functional pathways in the body. Glutamate deprivation leads to oxidant-induced apoptosis via selective activation of caspases and the activation of N-methyl-D-aspartate (NMDA) receptors.22 Excessive accumulation of glutamate, with subsequent overactivation of NMDA receptors, results in an increase in free calcium levels in the cytoplasm. An influx of calcium increases nitric oxide production and cell membrane lipid peroxidation, resulting in the overproduction of peroxynitrite.23 Peroxynitrite causes DNA damage and activates caspase-3.24 Mesna contains a sulfhydryl (SH) group and as such could prevent cell death induced by caspase activation.6 In addition to caspases, protein kinase C (PKC) and second mitochondria-derived activator of caspases (SMACs) are activated in apoptotic cells.6 PKC can play a role in apoptotic cell death. Thus, it is possible that mesna inhibits other apoptosisinducing pathways upstream of caspase-3 activation. A sufficient supply of extra- or intracellular SH groups may protect the neuron from destruction due to ischemic injury. In conclusion, we have shown that a single 150 mg/kg dose of mesna decreases caspase-3 activity in rabbit spinal cord subjected to I/R injury. However, mesna does not appear to be as neuroprotective as MPSS. Further studies will be necessary to determine whether mesna could be used to provide neuroprotection from apoptotic cell death after I/R injury in humans, and to determine what the optimal dose would be. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jocn.2009.07.108. References 1. Akdemir O, Berksoy I, Karaog˘lan A, et al. Therapeutic efficacy of Ac-DMQD-CHO, a caspase 3 inhibitor, for rat spinal cord injury. J Clin Neurosci 2008;15:672–8. 2. Okutan O, Solaroglu I, Beskonakli E, et al. Recombinant human erythropoietin decreases myeloperoxidase and caspase-3 activity and improves early functional results after spinal cord injury in rats. J Clin Neurosci 2007;14:364–8. 3. Solaroglu I, Kaptanoglu E, Okutan O, et al. Magnesium sulfate treatment decreases caspase-3 activity after experimental spinal cord injury in rats. Surg Neurol 2005;64:17–21. 4. Barut Sß, Ünlü AY, Karaog˘lan A, et al. The neuroprotective effects of z-DEVD.fmk, a caspase-3 inhibitor, on traumatic spinal cord injury in rats. Surg Neurol 2005;64:213–20. 5. Sener G, Sehirli O, Ercan F, et al. Protective effect of MESNA (2-mercaptoethane sulfonate) against hepatic ischemia/reperfusion injury in rats. Surg Today 2005;35:575–80. 6. Schwerdt G, Kirchhoff A, Freudinger R, et al. Mesna or cysteine prevents chloroacetaldehyde-induced cell death of human proximal tubule cells. Pediatr Nephrol 2007;22:798–803. 7. Lowry O, Rosenbrough N, Farr L, et al. Protein measurement with Folin phenol reagent. J Biol Chem 1951;182:265–75. 8. Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal death. A mitochondrial hypothesis. Stroke 1995;26:1478–89. 9. Sakurai M, Nagata T, Abe K, et al. 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;125:370–7. 10. Hayashi T, Sakuria M, Abe K, et al. Apoptosis of motor neurons with induction of caspases in the spinal cord after ischemia. Stroke 1998;29:1007–13. 11. Knoblach SM, Nikolaeva M, Huang X, et al. Multiple caspases are activated after traumatic brain injury: evidence for involvement in functional outcome. J Neurotrauma 2002;19:1155–70. 12. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 1982;11:491–8. 13. Citron BA, Arnold PM, Haynes NG, et al. Neuroprotective effects of caspase-3 inhibition on functional recovery and tissue sparing after acute spinal cord injury. Spine 2008;33:2269–77. 14. Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 1982;239:57–69. 15. Hearse DJ, Boli R. Reperfusion induced injury: manifestations, mechanisms, and clinical relevance. Cardiovasc Res 1992;26:101–8. 16. Li M, Ona VO, Chen M, et al. Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience 2000;99:333–42.
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