Sevoflurane Preconditioning Induces Protection To Spinal Cord Ischemia/reperfusion In Rabbits

【Background】Despite many strategies have been developed to increase the ischemic tolerance of spinal cord and minimize the incidence of neurological complications, spinal cord ischemia occurred in the patients undergoing thoracic aorta or thoracoabdominal aorta aneurysm surgery. The quoted figure 3% to 18% was incidence of residual permanent injury (acute and/or delayed paraplegia) caused by the ischemic episode. Spinal cord ischemia/reperfusion (I/R) injury could also take place during spinal surgery of tumor, disc decompression and deformity. The social expense associated with the medical management and lost earnings of this disorder over the course of one's life was significant.Sevoflurane, as a novel inhalational anesthetic with minimal pungency, low solubility, less hepatic toxicity, and absence of arrhythmogenicity is a preferable choice in clinic. Recently, studies demonstrated that sevoflurane preconditioning could induce ischemic tolerance in brain and heart, and alleviate hypoxic and ischemic injury. However, few study attempting to describe the protective effect of this agent on spinal cord I/R appeared.The present study was designed to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to spinal cord in a rabbit model of transient spinal cord ischemia, and the potential mechanism of the protection.【Methods】New Zealand White male rabbits (2.0 kg - 2.9 kg) were used in this experiment.Protocol 1: Animals were randomly assigned to 2 groups (n = 6, each). Animals in the I/R group received spinal cord I/R induced by infrarenal aorta occlusion for 20 min; Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining, TUNEL staining and p-ERK1/2 immunofluorescent staining.Protocol 2: Animals were randomly assigned to 4 groups (n = 10, each). Animals in the Sev+W1h group and Sev+W15min group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min 1 h or 15 min after preconditioning, animals were subjected to spinal cord I/R respectively. Animals in the O2+W1h group and O2+W15min group as control inhaled only 96% oxygen for 30 min instead. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for histopathological examination.Protocol 3: Animals were randomly assigned to 2 groups. Animals in the Sev group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min, while animals in the O2 group as control inhaled only 96% oxygen for 30 min instead. One hour after preconditioning, the animals were subjected to spinal cord I/R. Animals in each group were randomly assigned to 6 small groups by their end point of investigation (8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion, n = 6, each). All animals were assessed at 8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested for HE staining, Fluoro-Jade B staining and TUNEL staining.Protocol 4: Animals were randomly assigned to 4 groups (n = 8). U0126, an ERK1/2 inhibitor, was administered intravenously 20 min before the beginning of preconditioning in the U0126 + O2 and U0126 + Sev groups. DMSO was administered intravenously at the same time in the Vehicle + O2 and Vehicle + Sev groups. Animals were subjected to spinal cord I/R 1 h after preconditioning. The methods of preconditioning and spinal cord I/R procedure were the same as described in protocol 3. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining and TUNEL staining.Normal motor neurons, Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of the L5 spinal cord were counted.【Results】Protocol 1: Compared with Sham group, animals in the I/R group had lower neurological scores, less normal motor neurons, more Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of spinal cord (P < 0.01) 48 h after reperfusion. There were many p-ERK1/2-positive astroglias (double labled with GFAP, the marker of astroglia) in the white matter of spinal cord in I/R group, while all neurons were p-ERK1/2-negative. Protocol 2: 48 h after reperfusion, compared with O2+W1h group, animals in the Sev+W1h group had higher neurological scores and more normal motor neurons in the anterior of spinal cord (P < 0.01). There was no difference between the Sev+W15min group and O2+W15min group (P > 0.05).Protocol 3: From 1 d after reperfusion, animals in Sev group had higher neurological scores than those in O2 group (P < 0.05). There was no significant change of neurological scores from 2 d to 7 d after reperfusion in every group (P > 0.05). The number of normal neurons in the anterior of spinal cord decreased from 8 h after reperfusion in 2 groups, and downed to the bottom at 48 h after reperfusion, then the number had no significant change (P > 0.05). Animals in Sev group had more normal motor neurons than O2 group at every investigative time point (P < 0.05). The number of Fluoro-Jade B-positive neurons in the anterior of spinal cord increased from 8 h after reperfusion in Sev group, while it increased before 8 h after reperfusion in the O2 group, and rose to the top at 3 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less Fluoro-Jade B-positive neurons than O2 group at every investigative point except 7 d after reperfusion (P < 0.05). The number of TUNEL-positive neurons in the anterior of spinal cord elevated from 8 h after reperfusion in 2 groups, and rose to the top at 2 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less TUNEL-positive neurons than O2 group from 1 d to 5 d after reperfusion (P < 0.05).Protocol 4: Compared with Vehicle+Sev group, U0126+Sev group presented worse neurological outcomes, fewer viable neurons and more degenerative neurons and more apoptotic neurons (P≤0.01 for each comparison). There were no significant differences in the outcomes among Vehicle+O2, U0126+O2, and U0126+Sev groups (P > 0.05).【Conclusion】1. This study demonstrates that neuronal degeneration and apoptosis are important mechanisms of spinal cord I/R injury, as they presented within 5 d after spinal cord I/R.2. This experiment indicates that ERK1/2 plays a crucial role in spinal cord I/R, because there were many p-ERK1/2-positive astroglias in the white matter of spinal cord in animals suffered spinal cord I/R, while all neurons were negative staining.3. The investigation displays that 3.7% (1.0 MAC) sevoflurane preconditioning 1 h before ischemia induces rapid tolerance to spinal cord I/R in rabbits.4. The research proves that the protection of sevoflurane preconditioning is caused by attenuating neurological injury instead of delaying the injury. The beneficial effect could last 7 d after reperfusion, and there were no neuronal degeneration or apoptosis at that time.5. This test indicates that sevoflurane preconditioning induced rapid tolerance to spinal cord I/R in rabbits is possibly mediated through the activation of ERK1/2 and the pathway of ERK1/2 mediated for anti-apoptosis in the spinal cord.