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Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbits
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Life Sciences 82 (2008) 608 – 614 www.elsevier.com/locate/lifescie
Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbits Xiaojing Jiang a,b,⁎,1 , Enyi Shi c,1 , Liwen Li d , Yoshiki Nakajima a , Shigehito Sato a a
Department of Anesthesiology, Hamamatsu University School of Medicine, Hamamtsu, Japan Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, China c Department of Cardiac Surgery, First Affiliated Hospital, China Medical University, Shenyang, China d Department of Anesthesiology, Second Xiangya Hospital, Central South University, Changsha, China b
Received 23 August 2007; accepted 9 December 2007
Abstract Postconditioning can induce cardioprotection against ischemia. However, the data on postconditioning of the spinal cord is very limited. We investigated here whether co-application of ischemic preconditioning (IPC) and postconditioning can provide additive neuroprotection against prolonged spinal cord ischemia. Spinal cord ischemia was produced in rabbits by infrarenal aortic occlusion with a balloon catheter for 30 min. The four treatment groups were control (n = 10): no intervention; IPC (n = 10): a 5-minute aortic occlusion was performed 20 min before the prolonged ischemia; Postcon (n = 10): postconditioning comprised of four cycles of 1-minute occlusion/1-minute reperfusion was applied one minute after the start of reperfusion. IPC + postcon (n = 11): both IPC and postconditioning were applied. Functional evaluation with Tarlov score was performed during a 14-day observation period. Neurologic impairment was noticeably attenuated in the IPC + postcon group (compared with the control group, P b 0.01, at day 1, day 2, day 7 and day 14, respectively), but not in either the IPC or Postcon group. Plasma malondialdehyde levels after reperfusion were significantly decreased to a similar extent in the IPC, Postcon and IPC + Postcon groups (compared with the control group (P b 0.01). In the IPC + Postcon group, many more large motor neurons were preserved than in the control group (P b 0.05) and white matter injury was also markedly attenuated as evidenced by reduction of the vacuolation area of the white matter (P b 0.01) and decreased amyloid precursor protein immunoreactivity (P b 0.01). From this, we conclude that the combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injuries. © 2008 Elsevier Inc. All rights reserved. Keywords: Spinal cord; Ischemia–reperfusion; Preconditioning; Postconditioning
Introduction One of the most serious complications after surgical repair of descending and thoracoabdominal aortic aneurysms is paraplegia. In the published series, the incidence of paraplegia is still high, with values of 6.6% to 8.3% in patients with extent II thoracoabdominal aortic aneurysms (Coselli et al., 2000; Safi et al., 2003). Although the mechanisms of neurological injuries are still not fully ⁎ Corresponding author. Address: Department of Anesthesiology, First Affiliated Hospital, China Medical University, Nanjingbei Street 155#, Shenyang, China, 110001. Tel.: +86 24 23256666. E-mail addresses: [email protected], [email protected] (X. Jiang). 1 Drs. Xiaojing Jiang and Enyi Shi contributed equally to this work. 0024-3205/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.026
understood, the principal root of this complication is believed to be spinal cord ischemia. In addition, reperfusion with oxygenated blood after ischemia also has the potential to aggravate the spinal cord injury, an effect known as a reperfusion injury. Ischemic preconditioning (IPC), which was first described in 1986 as a paradoxical form of cardioprotection (Murry et al., 1986), has now been acknowledged to be a potent endogenous protection to enhance the tolerance against ischemia in several organs, including the spinal cord (Park et al., 2005; Zvara et al., 1999; Toumpoulis et al., 2004). Another endogenous form of cardioprotection, termed postconditioning, has recently been reported, in which a short series of repetitive cycles of brief reperfusion and reocclusion of the coronary artery were applied immediately at the onset of reperfusion, and this procedure has
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been shown to reduce myocardial injury to an extent comparable to that of IPC (Zhao et al., 2003). The cardioprotective effects induced by postconditioning have been further confirmed in different models and species (Darling et al., 2005; Tsang et al., 2004; Kin et al., 2005). Very recent studies showed that postconditioning also induced powerful neuroprotective effects. In a rat model of cerebral ischemia, postconditioning reduced infarct size and blocked apoptosis and free radical generation (Zhao et al., 2006). In our previous work, postconditioning attenuated neurologic injuries resulting from spinal cord ischemia, and the first few minutes of reperfusion were crucial to its neuroprotection (Jiang et al., 2006). Interestingly, although IPC and postconditioning produce similar cardioprotection, they intervene at opposite ends of the ischemic event. A combination of IPC and postconditioning induced additive cardioprotection in Yang's report (Yang et al., 2004), but failed in other models (Tsang et al., 2004; Halkos et al., 2004). It has not been reported whether sequential application of IPC and postconditioning can enhance the neuroprotective effects on ischemic spinal cords. In the current study, we compared the neuroprotective effects of IPC and postconditioning in a rabbit model subjected to a prolonged spinal cord ischemia and tested the hypothesis that co-application of IPC and postconditioning would provide additive neuroprotection compared to either intervention alone. Materials and methods Animals Japanese white rabbits weighing about 2 kg were used in the study. The animal protocol was approved by the Ethics Review Committee for Animal Experimentation at the Hamamatsu
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University School of Medicine and was in accordance with the NIH guidelines for use and care of laboratory animals. Surgical procedure Surgery was conducted according to the method described previously (Shi et al., 2007). Briefly, the rabbits were anesthetized with intravenous sodium pentobarbital (25 mg/kg). Core body temperature was continuously monitored with a rectal probe and was maintained at 38.5 ± 0.5 °C with the aid of a heating lamp. A 4F balloon-tipped catheter (Goodtec Inc, Huntington Beach, CA) was inserted through an arteriotomy in the left femoral artery and advanced 15 cm forward into the abdominal aorta. Preliminary investigations confirmed that the balloon should be positioned 0.5–1.2 cm distal to the left renal artery (Suzuki et al., 2005). After systemic heparinization (200 U/kg), spinal cord ischemia was induced by inflation of the balloon. Complete aortic occlusion was confirmed by reduction in distal aortic blood pressure to less than 20 mmHg, which was measured through the side hole of the balloon catheter. At the end of the operation, the catheter was removed and the femoral artery was reconstructed. Experimental protocol All rabbits were subjected to 30 min of spinal cord ischemia. The four treatment groups were as shown in Fig. 1: (1) control (n = 10): no intervention before ischemia or during reperfusion; (2) IPC (n = 10): a 5-minute spinal cord ischemia was performed 20 min before the prolonged spinal cord ischemia; (3) Postcon (n = 10): after 30 min of ischemia, full reperfusion was initiated for 1 min followed by four cycles of postconditioning (one cycle comprised a 1-minute occlusion and a 1-minute reperfusion); and
Fig. 1. Experimental groups and protocol. IPC = ischemic preconditioning; Postcon = postconditioning; IS = ischemia; R = reperfusion.
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(4) IPC + postcon (n = 11): the above IPC and postconditioning protocols were combined.
Statistical analysis
During a 14-day recovery after ischemia, hind-limb motor function was assessed by two observers blinded to the rabbits' treatment groups using the modified Tarlov scale (Tarlov, 1972): 0, no movement; 1, slight movement; 2, sit with assistance; 3, sit alone; 4, weak hop; and 5, normal hop.
Values are expressed as means ± standard deviation. The Kruskal–Wallis test was used for nonparametric values (Tarlov score, viable neurons and APP score) and the Mann–Whitney U test was used as a post-test to identify the specific differences between the groups. Parametric values were analyzed by oneway or two-way repeated-measures (time and group) analysis of variance followed by the Student–Newman–Keuls post hoc test. Statistical significance was defined as P b 0.05.
Determination of plasma malondialdehyde
Results
Plasma malondialdehyde (MDA), a marker of oxidantmediated lipid peroxidation, was quantified to estimate the extent of lipid peroxidation in the ischemic spinal cord (Gurcun et al., 2006). Arterial blood samples were collected at baseline, at the end of ischemia, and one hour and 24 h after reperfusion. MDA activity was measured using the Malondialdehyde Assay Kit (Northwest Life Science Specialties, Vancouver, WA) according to the procedure recommended by the manufacturer.
Physiologic parameters
Neurologic assessment
The physiological parameters are shown in Table 1. The mean blood pressure of distal aorta of the four groups was significantly decreased during the aortic occlusion (P b 0.01 vs baseline). There was no significant difference in the weights of the animals (P N 0.05). The rectal temperature and mean blood pressure of distal aorta were not significantly different among the four groups at any time point (P N 0.05).
Histological study Neurologic assessment All rabbits were sacrificed 14 days after spinal cord ischemia. Paraffin embedded sections (4 μm) of lumbar spinal cords (L4-6) were stained with hematoxylin and eosin. In cases where the cytoplasm was diffusely eosinophilic, the large motor neuron cells were considered to be “necrotic or dead.” When the cells demonstrated basophilic stippling (containing Nissl substance), the motor-neuron cells were considered to be “viable or alive” (Mutch et al., 1993). The intact motor neurons in the ventral gray matter were counted by a blinded investigator in three sections for each rabbit, and the results were then averaged. White matter injury was assessed by evaluation of the vacuolation in ventral, ventrolateral and lateral white matter using NIH imaging software. The percent area of vacuolation of the three target areas was calculated and the data were then averaged (Kurita et al., 2006). Immunohistochemical staining Immunohistochemical staining of amyloid precursor protein (APP) was used to label injured axons. Briefly, after deparaffinization, sections were blocked in normal serum and treated with the antibody against APP (Chemicon, Temecula, CA). Then, the sections were incubated with biotinylated secondary antibody followed by high sensitivity streptavidin conjugated to horseradish peroxidase (R&D System, Minneapolis, MN). Diaminobenzidine (DAB) was used as a chromogen for light microscopy. APP immunoreactivity was assessed in three areas of ventral, ventrolateral and lateral white matter with a dimension of 200 µm. Each area was divided into nine squares and a score of 0 (no APP accummulation) or 1 (APP accummulation in axonal swelling) was assigned to each square (Shi et al., 2007; Kurita et al., 2006). The total score of both sides in each animal was calculated (from 0 to 54).
The individual neurologic scores of the four groups 1, 2, 7 and 14 days after reperfusion are shown in Fig. 2. A 30-minute period of aortic occlusion resulted in severe lower-extremity neurologic deficits in the control group. Compared with the controls, rabbits of the IPC group and Postcon group had better motor function, but the differences did not reach statistical significance (P N 0.05 for each respective time point). However, sequential application of IPC and postconditioning noticeably enhanced motor function after spinal cord ischemia, with Tarlov
Table 1 Physiologic parameters Control (n = 10) IPC (n = 10) Postcon (n = 10) IPC + postcon (n = 11) Weight (kg)
2.2 ± 0.2
Rectal temperature (°C) Baseline 38.6 ± 0.2 Pre-ischemia 38.5 ± 0.3 Ischemia 38.3 ± 0.4 15 min Reperfusion 38.5 ± 0.3 15 min Mean distal aortic pressure Baseline 97 ± 6 Pre-ischemia 99 ± 5 Ischemia 16 ± 2* 15 min Reperfusion 95 ± 4 15 min
2.1 ± 0.2
2.2 ± 0.2
2.3 ± 0.2
38.6 ± 0.3 38.5 ± 0.4 38.2 ± 0.3
38.3 ± 0.4 38.4 ± 0.4 38.3 ± 0.3
38.3 ± 0.5 38.3 ± 0.4 38.1 ± 0.3
38.3 ± 0.5
38.6 ± 0.3
38.6 ± 0.2
100 ± 8 98 ± 4 17 ± 2*
104 ± 7 102 ± 5 16 ± 2*
101 ± 7 103 ± 6 17 ± 3*
100 ± 6
98 ± 4
98 ± 5
The values are expressed as means ± SD. *P b 0.01 vs baseline. IPC = ischemic preconditioning; Postcon = postconditioning.
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Fig. 2. Neurologic function assessed with Tarlov score at 1, 2, 7 and 14 days after spinal cord ischemia. Triangles represent individual rabbits. IPC = ischemic preconditioning; Postcon = postconditioning.
scores being significantly higher than those of the control group (P b 0.01 for each respective time point). Plasma MDA levels during ischemia and reperfusion There were no significant differences in plasma MDA levels among the four groups at baseline (Fig. 3). Infrarenal aorta occlusion induced a slight increase of MDA values with no statistical difference compared with the baseline. In the control group, the concentration of plasma MDA was significantly
increased at one hour after reperfusion, and a higher value was maintained until 24 h after reperfusion (P b 0.01 vs baseline). At these two time points, the plasma MDA levels of IPC, Postcon and IPC + postcon groups were remarkably lower than those of the control group (P b 0.01, respectively), but no significant difference was observed among the three intervention groups. MDA values at reperfusion at one hour in the IPC, Postcon, and IPC + Postcon groups were significantly lower compared to baseline (P b 0.01, respectively). Histological assessment
Fig. 3. Plasma malondialdehyde (MDA) levels during spinal cord ischemia and reperfusion. IPC = ischemic preconditioning; Postcon = postconditioning.
Representative sections of lumbar spinal cords stained with hematoxylin and eosin are shown in Fig. 4A and the results of viable motor neuron counting are summarized in Fig. 4B. A 30minute aortic occlusion induced severe neuronal damage in the animals of the control group 14 days after ischemia, as indicated by vacuolization, necrosis, and an almost total loss of motor neurons. In contrast, slighter histological changes were found in the lumbar spinal cords of the IPC + postcon group, and motor neurons were preserved intact to a much greater extent (P b 0.05, vs control group). Although more motor neurons also remained intact in the IPC and Postcon groups, the differences were not significant compared to the control group (P N 0.05). Vacuolation was apparent in the white matter of the control group, but was not so notable in the white matter of the IPC + postcon group, as shown in Fig. 5A. The percent area of
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Fig. 4. Histologic assessment of lumbar spinal cords 14 days after transient ischemia. A. Representative sections stained with hematoxylin and eosin (original magnification 100×). B. Number of large motor neurons in the ventral gray matter. IPC = ischemic preconditioning; Postcon = postconditioning.
Fig. 6. Expression of amyloid precursor protein (APP) in white matter of lumbar spinal cords. A. Photomicrographs of the immunohistochemical staining for APP in the ventrolateral white matter (original magnification 200×). B. APP scores of the four groups. IPC= ischemic preconditioning; Postcon = postconditioning.
vacuolation of the control group was significantly higher than that of the IPC + postcon group ( p b 0.01, Fig. 5B). Compared to the control group, the area of vacuolation was not noticably reduced in either the IPC group or Postcon group.
APP immunoreactivity Immunohistochemical staining revealed that APP was concentrated within axon bundles in the ventral, ventrolateral and lateral white matter 14 days after spinal cord ischemia. Representative photographs of immunohistochemical staining for APP in the ventrolateral region of white matter are shown in Fig. 6A. As summarized in Fig. 6B, only the APP scores of lumbar spinal cords of the IPC + postcon group, but not the IPC group or Postcon groups, were significantly lower than those of the control group (P b 0.01). Discussion
Fig. 5. Vacuolation in white matter of lumbar spinal cords. A. Representative photomicrographs of the ventrolateral white matter in hematoxylin and eosinstained sections (original magnification 200×). B. Percentage area of vacuolation in white matter. IPC= ischemic preconditioning; Postcon= postconditioning.
In the current study, we evaluated the neuroprotection conferred by IPC and postconditioning in a rabbit model subjected to prolonged spinal cord ischemia. The results demonstrated that sequential application of IPC and postconditioning effectively protected ischemic spinal cords, as evidenced by motor functional improvement and attenuation of histological damage of both gray matter and white matter. However, no powerful neuroprotection was detected when either IPC or postconditioning was performed alone. Protective efficiency of IPC against spinal cord ischemia has been shown in different models (Park et al., 2005; Zvara et al., 1999; Toumpoulis et al., 2004). The protection provided by IPC is dependent on the duration of ischemic pretreatment, the time window between the pretreatment and the subsequent insult, and the severity of the subsequent insult. In a similar rabbit model, 5minute IPC markedly attenuated the neurological injuries caused by 20 min of spinal cord ischemia (Park et al., 2005). The same
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episode of IPC was performed in the present study with a prolongation of ischemia to 30 min, and neuroprotection was attenuated to a lack of significance. In our previous work, postconditioning was demonstrated to protect ischemic spinal cords, in which a sequence of four cycles of 1-minute occlusion/1minute reperfusion applied one minute after 25-minute spinal cord ischemia was effective, and extending the number of the postconditioning cycles to six resulted in no significant gain in neuroprotection (Jiang et al., 2006). However, the 4-cycle postconditioning failed to induce significant neuroprotective effects in the current study, suggesting that the postconditioning may be protective for spinal cords only when the ischemic period is less than 30 min in a rabbit model. Although these two strategies conferred similar, non-significant neuroprotection, the combination of IPC and postconditioning was detected to provide robust neuroprotective effects. To our knowledge, this is the first report to speculate that combination of IPC and postconditioning can induce additive neuroprotection for ischemic spinal cords. It is still somewhat unclear whether protection of postconditioning and IPC can be enhanced by each other, since different studies have yielded mixed results. The combined protective effects of IPC and postconditioning did not appear to be additive in isolated perfused rat hearts (Tsang et al., 2004) and open-chest canines subjected to coronary occlusion (Halkos et al., 2004). However, in disagreement with these findings, Yang's group reported that coapplication of IPC and postconditioning further enhanced cardioprotection in an in vivo rabbit model (Yang et al., 2004). This discrepancy may be attributed to the different models of ischemia–reperfusion injury used. Collective studies confirm that reactive oxygen species (ROS) contribute to neuronal cell injuries secondary to ischemia and reperfusion (Chan, 2001). Upon the onset of reperfusion, there is a “respiratory burst” lasting several minutes that originates from a number of cellular sources. This oxidative burst is followed by a moderately, but persistently elevated production of oxygen radicals (Herkert et al., 2002). Oxygen radicals damage cellular lipids, proteins, and nucleic acids and initiate cell death signaling pathways after cerebral ischemia (Saito et al., 2005). Both IPC and postconditioning have been shown to reduce the generation of ROS in ischemic heart (Kevin et al., 2003; Kin et al., 2004), brain (Zhao et al., 2006), and spinal cord (Mutch et al., 1993), and the observed protection has been correlated with the inhibition of ROS generation. In this study, the lipid peroxidation products of spinal cords estimated by plasma MDA were comparably reduced by both IPC and postconditioning at one hour after reperfusion, and the reductions were maintained until 24 h later, indicating that the two interventions attenuate the large burst of ROS generation during the early moments of reperfusion, as well as sustained generation during late reperfusion. However, only equivalent MDA levels were detected when IPC and postconditioning were sequentially applied and there was no further decrease. Although we could not exclude that reduction of ROS generation contributed to the neuroprotection observed in the present study, combination of IPC and postconditioning must function by mechanisms other than inhibition of ROS generation. Some studies have demonstrated that IPC and
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postconditioning may share the same protein kinase cascades to induce their protection, including the PI3K-Akt and the MEK1/ 2-Erk1/2 pathways, although they function at the opposite ends of the ischemic event (Hausenloy and Yellon, 2006). Nonetheless, the additive neuroprotection of the current study strongly indicates that the mechanisms of action of IPC and postconditioning were different, which is consistent with Yang's conclusion (Yang et al., 2004). However, the mechanisms through which co-application of IPC and postconditioning induces additive neuroprotection against spinal cord ischemia remain to be defined in detail. In addition, we evaluated the white matter injury of spinal cords in the current study, which also contributed to motor dysfunction after spinal cord ischemia. Recent evidence shows that white matter injury is of equal importance to gray matter injury (Kanellopoulos et al., 2000; Follis et al., 1993), and white matter is even more vulnerable to ischemia than gray matter (Pantoni et al., 1996). The true extent of spinal cord injury may only be obtained with the assessment of both white and gray matter injuries (Follis et al., 1993). The white matter injury can be reflected by the vacuolation of white matter and accumulation of APP. The vacuolation indicates a segmental swelling of myelinated axons, the formation of spaces between myelin sheaths and axolemma, and astrocyte swelling (Kurita et al., 2006). APP is transported by fast anterograde axonal transport; therefore, the accumulation of APP at the sites of injury, accompanied by morphologic evidence of axonal damage in the form of axonal swelling or bulbs, has been regarded as evidence of axonal injury (Kurita et al., 2006). Our data demonstrate that combination of IPC and postconditioning attenuated white matter injury as well as gray matter injury resulting from spinal cord ischemia. In summary, the current study demonstrates for the first time that combination of IPC and postconditioning induces additive neuroprotective effects in a rabbit model of prolonged spinal cord ischemia, even though IPC or postconditioning alone failed to induce marked neuroprotection. Our observations provide compelling evidence for a novel strategy to protect spinal cords against ischemia–reperfusion injuries. The spinal cord ischemia discussed in the present study is a complication of surgery and the exact time of its occurrence can be known. Therefore, both IPC and postconditioning can readily be applied. We believe that sequential application of IPC and postconditioning has potential clinical value in the prevention of neurologic injuries after thoracic aneurysm surgery. Conclusion Combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injury. Acknowledgements This work was supported by a Grant-in-aid for Scientific Research (No. 18791075) from the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.
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Life Sciences 82 (2008) 608 – 614 www.elsevier.com/locate/lifescie
Co-application of ischemic preconditioning and postconditioning provides additive neuroprotection against spinal cord ischemia in rabbits Xiaojing Jiang a,b,⁎,1 , Enyi Shi c,1 , Liwen Li d , Yoshiki Nakajima a , Shigehito Sato a a
Department of Anesthesiology, Hamamatsu University School of Medicine, Hamamtsu, Japan Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, China c Department of Cardiac Surgery, First Affiliated Hospital, China Medical University, Shenyang, China d Department of Anesthesiology, Second Xiangya Hospital, Central South University, Changsha, China b
Received 23 August 2007; accepted 9 December 2007
Abstract Postconditioning can induce cardioprotection against ischemia. However, the data on postconditioning of the spinal cord is very limited. We investigated here whether co-application of ischemic preconditioning (IPC) and postconditioning can provide additive neuroprotection against prolonged spinal cord ischemia. Spinal cord ischemia was produced in rabbits by infrarenal aortic occlusion with a balloon catheter for 30 min. The four treatment groups were control (n = 10): no intervention; IPC (n = 10): a 5-minute aortic occlusion was performed 20 min before the prolonged ischemia; Postcon (n = 10): postconditioning comprised of four cycles of 1-minute occlusion/1-minute reperfusion was applied one minute after the start of reperfusion. IPC + postcon (n = 11): both IPC and postconditioning were applied. Functional evaluation with Tarlov score was performed during a 14-day observation period. Neurologic impairment was noticeably attenuated in the IPC + postcon group (compared with the control group, P b 0.01, at day 1, day 2, day 7 and day 14, respectively), but not in either the IPC or Postcon group. Plasma malondialdehyde levels after reperfusion were significantly decreased to a similar extent in the IPC, Postcon and IPC + Postcon groups (compared with the control group (P b 0.01). In the IPC + Postcon group, many more large motor neurons were preserved than in the control group (P b 0.05) and white matter injury was also markedly attenuated as evidenced by reduction of the vacuolation area of the white matter (P b 0.01) and decreased amyloid precursor protein immunoreactivity (P b 0.01). From this, we conclude that the combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injuries. © 2008 Elsevier Inc. All rights reserved. Keywords: Spinal cord; Ischemia–reperfusion; Preconditioning; Postconditioning
Introduction One of the most serious complications after surgical repair of descending and thoracoabdominal aortic aneurysms is paraplegia. In the published series, the incidence of paraplegia is still high, with values of 6.6% to 8.3% in patients with extent II thoracoabdominal aortic aneurysms (Coselli et al., 2000; Safi et al., 2003). Although the mechanisms of neurological injuries are still not fully ⁎ Corresponding author. Address: Department of Anesthesiology, First Affiliated Hospital, China Medical University, Nanjingbei Street 155#, Shenyang, China, 110001. Tel.: +86 24 23256666. E-mail addresses: [email protected], [email protected] (X. Jiang). 1 Drs. Xiaojing Jiang and Enyi Shi contributed equally to this work. 0024-3205/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.12.026
understood, the principal root of this complication is believed to be spinal cord ischemia. In addition, reperfusion with oxygenated blood after ischemia also has the potential to aggravate the spinal cord injury, an effect known as a reperfusion injury. Ischemic preconditioning (IPC), which was first described in 1986 as a paradoxical form of cardioprotection (Murry et al., 1986), has now been acknowledged to be a potent endogenous protection to enhance the tolerance against ischemia in several organs, including the spinal cord (Park et al., 2005; Zvara et al., 1999; Toumpoulis et al., 2004). Another endogenous form of cardioprotection, termed postconditioning, has recently been reported, in which a short series of repetitive cycles of brief reperfusion and reocclusion of the coronary artery were applied immediately at the onset of reperfusion, and this procedure has
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been shown to reduce myocardial injury to an extent comparable to that of IPC (Zhao et al., 2003). The cardioprotective effects induced by postconditioning have been further confirmed in different models and species (Darling et al., 2005; Tsang et al., 2004; Kin et al., 2005). Very recent studies showed that postconditioning also induced powerful neuroprotective effects. In a rat model of cerebral ischemia, postconditioning reduced infarct size and blocked apoptosis and free radical generation (Zhao et al., 2006). In our previous work, postconditioning attenuated neurologic injuries resulting from spinal cord ischemia, and the first few minutes of reperfusion were crucial to its neuroprotection (Jiang et al., 2006). Interestingly, although IPC and postconditioning produce similar cardioprotection, they intervene at opposite ends of the ischemic event. A combination of IPC and postconditioning induced additive cardioprotection in Yang's report (Yang et al., 2004), but failed in other models (Tsang et al., 2004; Halkos et al., 2004). It has not been reported whether sequential application of IPC and postconditioning can enhance the neuroprotective effects on ischemic spinal cords. In the current study, we compared the neuroprotective effects of IPC and postconditioning in a rabbit model subjected to a prolonged spinal cord ischemia and tested the hypothesis that co-application of IPC and postconditioning would provide additive neuroprotection compared to either intervention alone. Materials and methods Animals Japanese white rabbits weighing about 2 kg were used in the study. The animal protocol was approved by the Ethics Review Committee for Animal Experimentation at the Hamamatsu
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University School of Medicine and was in accordance with the NIH guidelines for use and care of laboratory animals. Surgical procedure Surgery was conducted according to the method described previously (Shi et al., 2007). Briefly, the rabbits were anesthetized with intravenous sodium pentobarbital (25 mg/kg). Core body temperature was continuously monitored with a rectal probe and was maintained at 38.5 ± 0.5 °C with the aid of a heating lamp. A 4F balloon-tipped catheter (Goodtec Inc, Huntington Beach, CA) was inserted through an arteriotomy in the left femoral artery and advanced 15 cm forward into the abdominal aorta. Preliminary investigations confirmed that the balloon should be positioned 0.5–1.2 cm distal to the left renal artery (Suzuki et al., 2005). After systemic heparinization (200 U/kg), spinal cord ischemia was induced by inflation of the balloon. Complete aortic occlusion was confirmed by reduction in distal aortic blood pressure to less than 20 mmHg, which was measured through the side hole of the balloon catheter. At the end of the operation, the catheter was removed and the femoral artery was reconstructed. Experimental protocol All rabbits were subjected to 30 min of spinal cord ischemia. The four treatment groups were as shown in Fig. 1: (1) control (n = 10): no intervention before ischemia or during reperfusion; (2) IPC (n = 10): a 5-minute spinal cord ischemia was performed 20 min before the prolonged spinal cord ischemia; (3) Postcon (n = 10): after 30 min of ischemia, full reperfusion was initiated for 1 min followed by four cycles of postconditioning (one cycle comprised a 1-minute occlusion and a 1-minute reperfusion); and
Fig. 1. Experimental groups and protocol. IPC = ischemic preconditioning; Postcon = postconditioning; IS = ischemia; R = reperfusion.
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(4) IPC + postcon (n = 11): the above IPC and postconditioning protocols were combined.
Statistical analysis
During a 14-day recovery after ischemia, hind-limb motor function was assessed by two observers blinded to the rabbits' treatment groups using the modified Tarlov scale (Tarlov, 1972): 0, no movement; 1, slight movement; 2, sit with assistance; 3, sit alone; 4, weak hop; and 5, normal hop.
Values are expressed as means ± standard deviation. The Kruskal–Wallis test was used for nonparametric values (Tarlov score, viable neurons and APP score) and the Mann–Whitney U test was used as a post-test to identify the specific differences between the groups. Parametric values were analyzed by oneway or two-way repeated-measures (time and group) analysis of variance followed by the Student–Newman–Keuls post hoc test. Statistical significance was defined as P b 0.05.
Determination of plasma malondialdehyde
Results
Plasma malondialdehyde (MDA), a marker of oxidantmediated lipid peroxidation, was quantified to estimate the extent of lipid peroxidation in the ischemic spinal cord (Gurcun et al., 2006). Arterial blood samples were collected at baseline, at the end of ischemia, and one hour and 24 h after reperfusion. MDA activity was measured using the Malondialdehyde Assay Kit (Northwest Life Science Specialties, Vancouver, WA) according to the procedure recommended by the manufacturer.
Physiologic parameters
Neurologic assessment
The physiological parameters are shown in Table 1. The mean blood pressure of distal aorta of the four groups was significantly decreased during the aortic occlusion (P b 0.01 vs baseline). There was no significant difference in the weights of the animals (P N 0.05). The rectal temperature and mean blood pressure of distal aorta were not significantly different among the four groups at any time point (P N 0.05).
Histological study Neurologic assessment All rabbits were sacrificed 14 days after spinal cord ischemia. Paraffin embedded sections (4 μm) of lumbar spinal cords (L4-6) were stained with hematoxylin and eosin. In cases where the cytoplasm was diffusely eosinophilic, the large motor neuron cells were considered to be “necrotic or dead.” When the cells demonstrated basophilic stippling (containing Nissl substance), the motor-neuron cells were considered to be “viable or alive” (Mutch et al., 1993). The intact motor neurons in the ventral gray matter were counted by a blinded investigator in three sections for each rabbit, and the results were then averaged. White matter injury was assessed by evaluation of the vacuolation in ventral, ventrolateral and lateral white matter using NIH imaging software. The percent area of vacuolation of the three target areas was calculated and the data were then averaged (Kurita et al., 2006). Immunohistochemical staining Immunohistochemical staining of amyloid precursor protein (APP) was used to label injured axons. Briefly, after deparaffinization, sections were blocked in normal serum and treated with the antibody against APP (Chemicon, Temecula, CA). Then, the sections were incubated with biotinylated secondary antibody followed by high sensitivity streptavidin conjugated to horseradish peroxidase (R&D System, Minneapolis, MN). Diaminobenzidine (DAB) was used as a chromogen for light microscopy. APP immunoreactivity was assessed in three areas of ventral, ventrolateral and lateral white matter with a dimension of 200 µm. Each area was divided into nine squares and a score of 0 (no APP accummulation) or 1 (APP accummulation in axonal swelling) was assigned to each square (Shi et al., 2007; Kurita et al., 2006). The total score of both sides in each animal was calculated (from 0 to 54).
The individual neurologic scores of the four groups 1, 2, 7 and 14 days after reperfusion are shown in Fig. 2. A 30-minute period of aortic occlusion resulted in severe lower-extremity neurologic deficits in the control group. Compared with the controls, rabbits of the IPC group and Postcon group had better motor function, but the differences did not reach statistical significance (P N 0.05 for each respective time point). However, sequential application of IPC and postconditioning noticeably enhanced motor function after spinal cord ischemia, with Tarlov
Table 1 Physiologic parameters Control (n = 10) IPC (n = 10) Postcon (n = 10) IPC + postcon (n = 11) Weight (kg)
2.2 ± 0.2
Rectal temperature (°C) Baseline 38.6 ± 0.2 Pre-ischemia 38.5 ± 0.3 Ischemia 38.3 ± 0.4 15 min Reperfusion 38.5 ± 0.3 15 min Mean distal aortic pressure Baseline 97 ± 6 Pre-ischemia 99 ± 5 Ischemia 16 ± 2* 15 min Reperfusion 95 ± 4 15 min
2.1 ± 0.2
2.2 ± 0.2
2.3 ± 0.2
38.6 ± 0.3 38.5 ± 0.4 38.2 ± 0.3
38.3 ± 0.4 38.4 ± 0.4 38.3 ± 0.3
38.3 ± 0.5 38.3 ± 0.4 38.1 ± 0.3
38.3 ± 0.5
38.6 ± 0.3
38.6 ± 0.2
100 ± 8 98 ± 4 17 ± 2*
104 ± 7 102 ± 5 16 ± 2*
101 ± 7 103 ± 6 17 ± 3*
100 ± 6
98 ± 4
98 ± 5
The values are expressed as means ± SD. *P b 0.01 vs baseline. IPC = ischemic preconditioning; Postcon = postconditioning.
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Fig. 2. Neurologic function assessed with Tarlov score at 1, 2, 7 and 14 days after spinal cord ischemia. Triangles represent individual rabbits. IPC = ischemic preconditioning; Postcon = postconditioning.
scores being significantly higher than those of the control group (P b 0.01 for each respective time point). Plasma MDA levels during ischemia and reperfusion There were no significant differences in plasma MDA levels among the four groups at baseline (Fig. 3). Infrarenal aorta occlusion induced a slight increase of MDA values with no statistical difference compared with the baseline. In the control group, the concentration of plasma MDA was significantly
increased at one hour after reperfusion, and a higher value was maintained until 24 h after reperfusion (P b 0.01 vs baseline). At these two time points, the plasma MDA levels of IPC, Postcon and IPC + postcon groups were remarkably lower than those of the control group (P b 0.01, respectively), but no significant difference was observed among the three intervention groups. MDA values at reperfusion at one hour in the IPC, Postcon, and IPC + Postcon groups were significantly lower compared to baseline (P b 0.01, respectively). Histological assessment
Fig. 3. Plasma malondialdehyde (MDA) levels during spinal cord ischemia and reperfusion. IPC = ischemic preconditioning; Postcon = postconditioning.
Representative sections of lumbar spinal cords stained with hematoxylin and eosin are shown in Fig. 4A and the results of viable motor neuron counting are summarized in Fig. 4B. A 30minute aortic occlusion induced severe neuronal damage in the animals of the control group 14 days after ischemia, as indicated by vacuolization, necrosis, and an almost total loss of motor neurons. In contrast, slighter histological changes were found in the lumbar spinal cords of the IPC + postcon group, and motor neurons were preserved intact to a much greater extent (P b 0.05, vs control group). Although more motor neurons also remained intact in the IPC and Postcon groups, the differences were not significant compared to the control group (P N 0.05). Vacuolation was apparent in the white matter of the control group, but was not so notable in the white matter of the IPC + postcon group, as shown in Fig. 5A. The percent area of
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Fig. 4. Histologic assessment of lumbar spinal cords 14 days after transient ischemia. A. Representative sections stained with hematoxylin and eosin (original magnification 100×). B. Number of large motor neurons in the ventral gray matter. IPC = ischemic preconditioning; Postcon = postconditioning.
Fig. 6. Expression of amyloid precursor protein (APP) in white matter of lumbar spinal cords. A. Photomicrographs of the immunohistochemical staining for APP in the ventrolateral white matter (original magnification 200×). B. APP scores of the four groups. IPC= ischemic preconditioning; Postcon = postconditioning.
vacuolation of the control group was significantly higher than that of the IPC + postcon group ( p b 0.01, Fig. 5B). Compared to the control group, the area of vacuolation was not noticably reduced in either the IPC group or Postcon group.
APP immunoreactivity Immunohistochemical staining revealed that APP was concentrated within axon bundles in the ventral, ventrolateral and lateral white matter 14 days after spinal cord ischemia. Representative photographs of immunohistochemical staining for APP in the ventrolateral region of white matter are shown in Fig. 6A. As summarized in Fig. 6B, only the APP scores of lumbar spinal cords of the IPC + postcon group, but not the IPC group or Postcon groups, were significantly lower than those of the control group (P b 0.01). Discussion
Fig. 5. Vacuolation in white matter of lumbar spinal cords. A. Representative photomicrographs of the ventrolateral white matter in hematoxylin and eosinstained sections (original magnification 200×). B. Percentage area of vacuolation in white matter. IPC= ischemic preconditioning; Postcon= postconditioning.
In the current study, we evaluated the neuroprotection conferred by IPC and postconditioning in a rabbit model subjected to prolonged spinal cord ischemia. The results demonstrated that sequential application of IPC and postconditioning effectively protected ischemic spinal cords, as evidenced by motor functional improvement and attenuation of histological damage of both gray matter and white matter. However, no powerful neuroprotection was detected when either IPC or postconditioning was performed alone. Protective efficiency of IPC against spinal cord ischemia has been shown in different models (Park et al., 2005; Zvara et al., 1999; Toumpoulis et al., 2004). The protection provided by IPC is dependent on the duration of ischemic pretreatment, the time window between the pretreatment and the subsequent insult, and the severity of the subsequent insult. In a similar rabbit model, 5minute IPC markedly attenuated the neurological injuries caused by 20 min of spinal cord ischemia (Park et al., 2005). The same
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episode of IPC was performed in the present study with a prolongation of ischemia to 30 min, and neuroprotection was attenuated to a lack of significance. In our previous work, postconditioning was demonstrated to protect ischemic spinal cords, in which a sequence of four cycles of 1-minute occlusion/1minute reperfusion applied one minute after 25-minute spinal cord ischemia was effective, and extending the number of the postconditioning cycles to six resulted in no significant gain in neuroprotection (Jiang et al., 2006). However, the 4-cycle postconditioning failed to induce significant neuroprotective effects in the current study, suggesting that the postconditioning may be protective for spinal cords only when the ischemic period is less than 30 min in a rabbit model. Although these two strategies conferred similar, non-significant neuroprotection, the combination of IPC and postconditioning was detected to provide robust neuroprotective effects. To our knowledge, this is the first report to speculate that combination of IPC and postconditioning can induce additive neuroprotection for ischemic spinal cords. It is still somewhat unclear whether protection of postconditioning and IPC can be enhanced by each other, since different studies have yielded mixed results. The combined protective effects of IPC and postconditioning did not appear to be additive in isolated perfused rat hearts (Tsang et al., 2004) and open-chest canines subjected to coronary occlusion (Halkos et al., 2004). However, in disagreement with these findings, Yang's group reported that coapplication of IPC and postconditioning further enhanced cardioprotection in an in vivo rabbit model (Yang et al., 2004). This discrepancy may be attributed to the different models of ischemia–reperfusion injury used. Collective studies confirm that reactive oxygen species (ROS) contribute to neuronal cell injuries secondary to ischemia and reperfusion (Chan, 2001). Upon the onset of reperfusion, there is a “respiratory burst” lasting several minutes that originates from a number of cellular sources. This oxidative burst is followed by a moderately, but persistently elevated production of oxygen radicals (Herkert et al., 2002). Oxygen radicals damage cellular lipids, proteins, and nucleic acids and initiate cell death signaling pathways after cerebral ischemia (Saito et al., 2005). Both IPC and postconditioning have been shown to reduce the generation of ROS in ischemic heart (Kevin et al., 2003; Kin et al., 2004), brain (Zhao et al., 2006), and spinal cord (Mutch et al., 1993), and the observed protection has been correlated with the inhibition of ROS generation. In this study, the lipid peroxidation products of spinal cords estimated by plasma MDA were comparably reduced by both IPC and postconditioning at one hour after reperfusion, and the reductions were maintained until 24 h later, indicating that the two interventions attenuate the large burst of ROS generation during the early moments of reperfusion, as well as sustained generation during late reperfusion. However, only equivalent MDA levels were detected when IPC and postconditioning were sequentially applied and there was no further decrease. Although we could not exclude that reduction of ROS generation contributed to the neuroprotection observed in the present study, combination of IPC and postconditioning must function by mechanisms other than inhibition of ROS generation. Some studies have demonstrated that IPC and
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postconditioning may share the same protein kinase cascades to induce their protection, including the PI3K-Akt and the MEK1/ 2-Erk1/2 pathways, although they function at the opposite ends of the ischemic event (Hausenloy and Yellon, 2006). Nonetheless, the additive neuroprotection of the current study strongly indicates that the mechanisms of action of IPC and postconditioning were different, which is consistent with Yang's conclusion (Yang et al., 2004). However, the mechanisms through which co-application of IPC and postconditioning induces additive neuroprotection against spinal cord ischemia remain to be defined in detail. In addition, we evaluated the white matter injury of spinal cords in the current study, which also contributed to motor dysfunction after spinal cord ischemia. Recent evidence shows that white matter injury is of equal importance to gray matter injury (Kanellopoulos et al., 2000; Follis et al., 1993), and white matter is even more vulnerable to ischemia than gray matter (Pantoni et al., 1996). The true extent of spinal cord injury may only be obtained with the assessment of both white and gray matter injuries (Follis et al., 1993). The white matter injury can be reflected by the vacuolation of white matter and accumulation of APP. The vacuolation indicates a segmental swelling of myelinated axons, the formation of spaces between myelin sheaths and axolemma, and astrocyte swelling (Kurita et al., 2006). APP is transported by fast anterograde axonal transport; therefore, the accumulation of APP at the sites of injury, accompanied by morphologic evidence of axonal damage in the form of axonal swelling or bulbs, has been regarded as evidence of axonal injury (Kurita et al., 2006). Our data demonstrate that combination of IPC and postconditioning attenuated white matter injury as well as gray matter injury resulting from spinal cord ischemia. In summary, the current study demonstrates for the first time that combination of IPC and postconditioning induces additive neuroprotective effects in a rabbit model of prolonged spinal cord ischemia, even though IPC or postconditioning alone failed to induce marked neuroprotection. Our observations provide compelling evidence for a novel strategy to protect spinal cords against ischemia–reperfusion injuries. The spinal cord ischemia discussed in the present study is a complication of surgery and the exact time of its occurrence can be known. Therefore, both IPC and postconditioning can readily be applied. We believe that sequential application of IPC and postconditioning has potential clinical value in the prevention of neurologic injuries after thoracic aneurysm surgery. Conclusion Combination of IPC and postconditioning induces additive neuroprotective effects for spinal cord against ischemia and reperfusion injury. Acknowledgements This work was supported by a Grant-in-aid for Scientific Research (No. 18791075) from the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.
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