Neuroprotection against Spinal Cord Ischemia–Reperfusion Injury Induced by Different Ischemic Postconditioning Methods: Roles of Phosphatidylinositol 3-Kinase–Akt and Extracellular Signal–regulated Kinase

Male Japanese white rabbits weighing 1.8–2.5 kg were used in the study. The animal protocol was approved by the Ethics Review Committee for Animal Experimentation of China Medical University (Shenyang, P. R. China) and was in accordance with the Guide for the Use and Care of Laboratory Animals  (National Institutes of Health, Bethesda, Maryland).

Surgery was conducted according to the method described previously.16Briefly, 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 22-gauge catheter was inserted into the ear artery to measure proximal blood pressure. A 4-French balloon-tipped catheter (Goodtec Inc., Huntington Beach, CA) was inserted through an arteriotomy in the left femoral artery and advanced 15 cm retrograde into the abdominal aorta. Preliminary investigations confirmed that the balloon should be positioned 0.5 to 1.2 cm distal to the left renal artery.21After 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. Arterial blood gas analysis and blood glucose were recorded before ischemia (baseline), 15 min after ischemia, and 20 min after reperfusion.

Before the balloon catheter was placed, the intervertebral space between L5 and L6 was punctured with a 16-gauge needle, and polyethylene-10 tubing was inserted through it into the subarachnoid space. The desired position of the catheter was confirmed by cautious aspiration of cerebrospinal fluid. After intrathecal injection of vehicle or the drugs, the catheter was removed.22 

All rabbits except the sham animals were subjected to 25 min of spinal cord ischemia. Six rabbits were assigned randomly to each of the following 10 groups, as shown in figure 1:

1. Sham group: Rabbits underwent the same surgical procedure and intrathecal injection of 10% dimethyl sulfoxide (DMSO), 20 μl, without aortic occlusion.

2. Control, LY (LY-294002), or PD (PD-98059) groups: Intrathecal injection of 10% DMSO, 20 μl; PI3K–Akt inhibitor LY-294002, 20 μl (0.1% in 10% DMSO); or ERK inhibitor PD-98059, 20 μl (0.015% in 10% DMSO), was conducted 1 min before the full reperfusion.

3. Postcon A (standard postconditioning), Postcon A + LY, Postcon A + PD: After 25 min of ischemia, full reperfusion was initiated for 1 min followed by 5 cycles of postconditioning (one cycle comprised 1-min occlusion and 1-min reperfusion). Intrathecal injection of DMSO, 20 μl; LY-294002, 20 μl (0.1% in 10% DMSO); or PD-98059, 20 μl (0.015% in 10% DMSO), was conducted 1 min before the start of reperfusion.

4. Postcon B (modified postconditioning), Postcon B + LY, Postcon B + PD: During the first 10 min of reperfusion, blood flow was partially restored and the mean blood pressure of the distal aorta was controlled between 45 and 55 mmHg by adjusting the volume of the balloon. Then, complete reperfusion was performed. Intrathecal injection of DMSO, 20 μl; LY-294002, 20 μl (0.1% in 10% DMSO); or PD-98059, 20 μl (0.015% in 10% DMSO), was conducted 1 min before the partial reperfusion.

During a 28-day recovery after ischemia, hind-limb motor function was assessed by two blinded observers using a modified Tarlov scale23: 0, paraplegic with no evident lower extremity motor function; 1, poor lower extremity motor function, weak antigravity movement only; 2, moderate lower extremity function with good antigravity strength but inability to draw legs under body; 3, excellent motor function with the ability to draw legs under body and hop, but not normally; and 4, normal motor function.

For histologic study, rabbits were killed 28 days after the transient spinal cord ischemia. Paraffin-embedded sections (4 μm) of lumbar spinal cords (L4–L6) were stained with hematoxylin–eosin. In cases in which 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.24The 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.

In a parallel series of experiments, spinal cords were collected 2 h after reperfusion from the following groups: control, Postcon A, Postcon B, LY, Postcon A + LY, Postcon B + LY, PD, Postcon A + PD, and Postcon B + PD (n = 4/group). Total Akt, phosphorylation states of Akt (phospho-Akt), total ERK, and phosphorylation states of ERK (phospho-ERK) proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis using specific antibodies as described previously.25The optical density for each band on the Western blot was quantified using the National Institutes of Health image program and was normalized to the corresponding Ponceau stain signal.

Parametric values are reported as mean ± SD. The Kruskal–Wallis test was used for nonparametric values (Tarlov score and number of neurons), and the Mann–Whitney U test was used as a posttest to identify the specific differences between the groups. Parametric values were analyzed by one-way 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 < 0.05.

A total of 64 rabbits were enrolled in the current study. Two rabbits were excluded as a result of technical difficulties with the preparation of spinal cord ischemia model. Another 2 rabbits were omitted because of death 1 and 4 days after the surgery.

Table 1indicates the physiologic parameters from all the groups. The mean blood pressure of distal aorta of all the groups except the sham group was significantly decreased during the aortic occlusion (P < 0.01 vs.  baseline). There were no significant differences in body weight, hemodynamics, rectal temperature, blood gas analysis data, or blood glucose among the groups at any time point (P > 0.05).

The individual neurologic scores of the 10 groups 1, 3, 7, and 28 days after the operation are shown in figure 2. The sham animals retained a normal motor function of lower limbs throughout the observation period. A 25-min aortic occlusion resulted in severe lower extremity neurologic deficits in the control rabbits, whereas the two postconditioning methods remarkably enhanced the motor function of the lower limbs after spinal cord ischemia as indicated by the significantly higher Tarlov scores of the Postcon A and Postcon B groups at the four observation time points (P < 0.05 vs.  control group). Equivalent Tarlov scores were observed between the Postcon A group and Postcon B group at the four observation time points (P > 0.05). The neuroprotective effects induced by the two postconditioning methods were completely abolished by intrathecal injection of LY-294002 or PD-98059. The Tarlov scores of Postcon A + LY and Postcon A + PD were significantly lower than those of Postcon A (P < 0.05). The Tarlov scores of Postcon B + LY and Postcon B + PD were significantly lower than those of Postcon B (P < 0.05). No significant differences were detected between the Tarlov scores of the control group with those of the LY group or PD group at each time point (P > 0.05).

Representative sections of lumbar spinal cords stained with hematoxylin–eosin are shown in figure 3A, and the results of counting viable motor neurons are summarized in figure 3B. In the sham-operated animals, the spinal cord was intact, and many large motor neurons were present in the anterior horn. A 25-min aortic occlusion induced severe neurologic damage in animals of the control group 28 days after ischemia, as indicated by vacuolization, frank necrosis, and an almost total loss of motor neurons. Similar histologic damages were found in the following groups: LY, PD, Postcon A + LY, Postcon A + PD, Postcon B + LY, and Postcon B + PD. There were no significant differences in the intact motor neuron count of these groups compared with that of the control group (P > 0.05). In contrast, slighter histologic changes were detected in lumbar spinal cords of animals in the Postcon A and Postcon B groups, and the intact motor neurons were preserved to a much greater extent (P < 0.05 vs.  control group). No significant difference in the number of motor neurons was detected between the Postcon A and Postcon B groups (P > 0.05).

Compared with the control animals, the two postconditioning methods resulted in a significantly higher expression of phospho-Akt, but not total Akt (in arbitrary units, Postcon A 61.7 ± 8.6, Postcon B 67.8 ± 11.6 vs.  control 44.0 ± 9.6; P < 0.05). No significant difference in the expression of phospho-Akt was detected between the Postcon A and Postcon B groups (in arbitrary units, 61.7 ± 8.6 vs.  67.8 ± 11.6; P > 0.05). The increased expressions of phospho-Akt induced by the two postconditioning methods were completely abolished in the presence of PI3K inhibitor LY-294002 (in arbitrary units, Postcon A + LY 42.3 ± 16.0, Postcon B + LY 36.3 ± 12.1 vs.  control 44.0 ± 9.6; P > 0.05). A representative Western blot image and the results of densitometric analysis are depicted in figure 4.

As illustrated in figure 5, the expressions of phospho-ERK, but not total ERK, were also markedly enhanced by the two postconditioning methods to an equivalent level, which was much higher than that of the control group (in arbitrary units, Postcon A 71.7 ± 6.9, Postcon B 74.8 ± 12.1 vs.  control 53.0 ± 11.2; P < 0.05). Intrathecal injection of ERK inhibitor PD-98059 inhibited the phospho-ERK–increasing effects afforded by the two postconditioning methods (in arbitrary units, Postcon A + PD 49.8 ± 12.0, Postcon B + PD 39.3 ± 9.1 vs.  control 53.0 ± 11.2; P > 0.05).

The salient findings of the current study can be summarized as follows: (1) Partial restoration of the blood flow at the first 10 min of reperfusion by control of the perfusion pressure to 45–55 mmHg, which was regarded as modified ischemic postconditioning, protected the spinal cord against ischemia–reperfusion injury to an extent comparable to standard ischemic postconditioning. (2) The neuroprotective effects induced by the two postconditioning methods were abolished by pharmacologic inhibition of either PI3K–Akt or ERK. (3) The two postconditioning methods significantly enhanced the expressions of both phospho-Akt and phospho-ERK in the spinal cord.

Timely restoration of blood flow is believed to be the most effective treatment to salvage neural tissue from prolonged ischemia. However, there is convincing evidence that sudden and full restoration of blood flow to ischemic cerebral tissue may paradoxically exaggerate injury that is not present at the end of ischemia. In clinical trials, patients treated with thrombolytic therapy have shown a 6% rate of intracerebral hemorrhage, which was balanced against a 30% improvement in functional outcome over controls. Destruction of the microvasculature and extension of the infarct area occur after cerebral reperfusion.26,27Ischemic postconditioning initially referred to a stuttering reperfusion performed immediately after reperfusion, for preventing reperfusion injury in myocardium. Thereafter, collective studies provided insights into the protective effects of postconditioning on cerebral ischemia. In rat models of focal cerebral ischemia, ischemic postconditioning was found to reduce infarct size12–14and inhibit neuronal apoptosis.11It has evolved into a concept that ischemic postconditioning may be a novel avenue to protect against brain injury after stroke.28In a rabbit model, we first reported the neuroprotective effects of postconditioning against ischemia–reperfusion injury of spinal cords.15The neuroprotective effects of ischemic postconditioning can be mainly attributed to reduction of reperfusion injury by the avoidance of sudden and full restoration of reperfusion. To attenuate the reperfusion injury, staged or controlled reperfusion with retarded restoration of full coronary blood flow or perfusion pressure has been proposed for many years.29Gradual reperfusion was demonstrated to induce neuroprotection on ischemic brain.12Controlled low-pressure perfusion was tested in our previous work and was found to protect ischemic spinal cords.16Just as the standard ischemic postconditioning, the protective intervention of this study was also performed at the beginning of reperfusion to avoid the sudden and full restoration of blood supply; therefore, we regarded this method as modified ischemic postconditioning. In a study by Bopassa et al. ,30low-pressure perfusion was confirmed to generate cardioprotection as robust as that of postconditioning. However, it is not known whether low-pressure perfusion, modified postconditioning, is also as powerful as standard postconditioning to protect ischemic spinal cords. Our previous results showed that four to six cycles of 1-min occlusion and 1-min reperfusion applied at the just beginning of reperfusion were neuroprotective against spinal cord injury in rabbits.15In the current study, five cycles of 1-min occlusion and 1-min reperfusion was chosen, and the total period of postconditioning was 10 min. Hence, the two postconditioning methods were applied in the same model, and the total intervention times were equivalent. As expected, similar neuroprotective effects were detected as evidenced by the improvement of motor function and reduction of histologic damage in spinal cords. The results indicate that controlled low-pressure perfusion at the beginning of reperfusion, modified postconditioning, possesses neuroprotective effects against ischemia–reperfusion injury of the spinal cord comparable to that of standard postconditioning. Unlike standard postconditioning, the modified method can be performed without additional ischemia, which may be more acceptable for clinicians.

Although the current study has not uncovered the full mechanisms of the neuroprotective effects of postconditioning, some elements, including PI3K–Akt and ERK, have been identified in the signal transduction pathway. Several studies have confirmed that postconditioning protected the ischemic heart by activating the PI3K–Akt and/or ERK cascades.10,17In subsequent studies, the role of the P13K–Akt pathway has received major attention in different models of brain ischemic postconditioning. In vivo  and in vitro  data demonstrated that the infarction-reducing effects of postconditioning were abrogated in the presence of PI3K inhibitor LY294002.13In another rat model of focal cerebral ischemia, PI3K was detected to contribute to the long-term protection of postconditioning against stroke in rats.12Beneficial effects of ischemic postconditioning on global cerebral ischemia and reperfusion-induced behavioral deficits in mice were also confirmed to be mediated by activation of PI3K pathway.18In the current study, the PI3K–Akt pathway was activated, as the Akt was markedly phosphorylated by the two postconditioning methods. This phosphorylation, commensurate with the functional and structural protection, was abrogated by administration of PI3K inhibitor LY294002. Hence, our data suggest that the PI3K–Akt pathway is the predominant mediator of the neuroprotection induced by both standard and modified postconditioning.

However, while the role played by phosphorylated Akt in mediating neuroprotection has been sufficiently elucidated in different models of cerebral ischemia as well as spinal cord ischemia reported in the current study, contrasting results have been obtained with phosphorylated ERK. ERK activation has been shown to be involved in the mechanism of ischemic tolerance in the rat hippocampus19and the neuroprotection induced by ischemic preconditioning,13whereas in some other studies, inhibition of ERK has been indicated to be crucial in mediating neuroprotection induced by some neuroprotective agents.31ERK also contributed to neural cell death in the animal models of ischemia- and trauma-induced brain injuries.32Still now, little is known about the function of ERK in the neuroprotection of postconditioning. In the current study, the two postconditioning strategies markedly enhanced the expression of phospho-ERK in the spinal cord, and their neuroprotection was abrogated with intrathecal injection of ERK-selective antagonist, suggesting that the ERK pathway is required for sustained spinal cord protection of postconditioning.

Therefore, our pharmacologic studies reveal that the spinal cord protection observed in the current study is mediated by both PI3K and ERK pathways. Expressions of both PI3K and ERK were shown to prolong phosphorylation in the cortex of posctconditioned rats; however, different from our results, the neuroprotection after postconditioning was inhibited only in the presence of LY294002, which blocks Akt activation, but not U0126, the ERK inhibitor.13In accord with our results, it has been reported that PI3K combined with ERK mediated the neuroprotective effects afforded by ischemic preconditioning,13MCI-186,33brain-derived neurotrophic factor,34erythropoietin,35and postischemic administration of orthovanadate.36In myocardial protection generated by postconditioning, the activation of PI3K and/or ERK may converge on the mitochondrial permeability transition pore.37Evidence obtained over the past two decades shows that reactive oxygen species are involved in brain lesions due to cerebral ischemia–reperfusion. The mitochondria are the primary intracellular source of reactive oxygen species, because they generate huge numbers of oxidative-reduction reactions and use massive amounts of oxygen.38Therefore, the spinal cord protection of postconditioning may also be mediated by inhibition of the mitochondrial permeability transition pore through the activation of PI3K and ERK. However, further studies are required to delineate the downstream factors of PI3K and ERK in the postconditioning-mediated protection on the spinal cord.

In conclusion, the current study provides novel insights into the spinal cord protection induced by standard postconditioning, a series of brief mechanical interruptions of reperfusion at onset of reperfusion, and modified postconditioning, partial restoration of blood flow by controlled low-pressure perfusion at the beginning of reperfusion. Our findings demonstrate for the first time that the two postconditioning methods induce neuroprotective effects on the spinal cord to a comparable extent and share a common molecular mechanism, in which PI3K and ERK pathways play crucial roles. As a very simple procedure, postconditioning afforded by the two methods possesses a potential clinical value in the prevention of neurologic injury after thoracic aneurysm surgery.