Oxidative Stress as Secondary Injury Mechanism After Mechanical Trauma in the Spinal Cord



Fig. 18.1
Change with time in the ICAM-1 mRNA/G3PDH mRNA ratio after compression injury. The relative amount of ICAM-1 mRNA was quantified by densitometry and expressed as the lCAM-1/G3PDH ratio. Points and bars show the mean ± SD of values obtained in three rats at each determination point. The ratio increased after injury, and the amount of increase was bigger in severe injury [13]



MPO increased after SCI by 688.9 % at 4 h and 476.7 % at 24 h. In the mild incomplete injury model (25-g weight for 5 mm), intravenous administration of ICAM-1 mAb (1 mg/kg; ICAM-l-mAb-treated group) 30 mm after significantly inhibited the increase of MPO at 4 and 24 h after injury by 43.0 % and 26.6 %, respectively. The injury caused spinal cord edema and water content of the cord increased by 2.7 %, and 1CAM-1 mAb treatment significantly reduced this edema by 1.1 %. After SCI, SCBF measured by H2 clearance method decreased from 45.2 ± 3.5 before injury to 31.0 ± 3.5 mL/min/100 g at 1 h, 30.5 ± 5.1 mL/min/100 g at 2 h, and 29.4 ± 5.6 mL/min/100 g at 4 h. The decrease of SCBF was partially prevented by ICAM-l mAb injection; it was 39.9 ± 6.0 mL/min/l00 g at 1 h (p < 0.05), but not at 2 h (32.3 ± 3.4 mL/min/l00 g) or 4 h (30.8 ± 3.4 mL/min/l00 g) after SCI.

Motor function was evaluated using several parameters. In the control group injected saline, motor dysfunction increased in the first 24 h after SCI as assessed by Tarlov’s motor scale and the inclined plane test, the deterioration of motor function was not observed in the ICAM-1-mAb-treated group. The ICAM-l-mAb-treated group showed significantly better recovery than the control group throughout 3 weeks after injury (Fig. 18.2a, b). The footprint analysis before and 3 weeks after SCI showed that the distance between the central pads of the hindfeet increased significantly by 73.4 %, and the stride lengths of the right hind limb decreased significantly by 34.6 % after SCI in the control group, but did not change significantly in the ICAM-l-mAb-treated group (Fig. 18.3). Joint angles during swing (swing) and at the beginning of stance (beginning stance) decreased 34.3 % and 12.0 %, respectively, in the control rats, but not in ICAM-l-mAb-treated rats.

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Fig. 18.2
Effect of ICAM-1 mAb treatment on the hind limb motor function after compression of the spinal cord for 5 min with a 25-g weight. ICAM-1 mAb (1 mg/kg) was injected via the tail vein 30 min after injury. Data are mean ± SEM values of Tarlov’s motor scale and mean ± SD values of the other analysis for 12 rats. The degree of motor function was assessed every day for 21 days after injury according to Tarlov’s criteria (a) and the inclined plane test (b). Tarlov’s motor scale is as follows: 0, no voluntary movement; 1, perceptible movement of joints; 2, good movement of joints but inability to stand; 3, ability to stand and walk; 4, complete recovery. In the inclined plane test, the maximum inclination of the plane, at which rats could maintain themselves for 5 s. *Values for the ICAM-1-mAb-treated group were significantly different from those for controls (Mann–Whitney U-test in Tarlov’s criteria and Student’s unpaired t-test in the inclined plane test) [13]


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Fig. 18.3
Footprint analysis 3 weeks after injury. The base of support was determined by measuring the distance between the central pads of the hind paws. The stride lengths of the right and left hind paws were measured in two consecutive prints. A significant increase in the base of support and a significant decrease in the right stride length were observed in the saline-treated group, but not in the ICAM-1-mAb-treated group. *Significantly different from normal and ICAM-1-mAb-treated rats (Student’s unpaired t-test) [13]




18.3 Nitric Oxide [18]


Nitric oxide (NO) was found to be an endothelium-derived relaxing factor [19]. In the central nervous system, NO may be neurotoxic by causing overstimulation of the N-methyl-n-aspartate receptor or cause hypotension and hyperalgesia and may be neuroprotective through anti-inflammatory or antioxidant effects [20–22]. We measured the expression of NO and NO synthase (NOS) after mechanical trauma in the spinal cord. Its roles in the secondary injury processes were investigated by evaluating the effects of an NOS inhibitor at different time point of mechanical trauma.


18.3.1 Expression of NO and Its Correlation with Oxidative Stress


Because of its short life in vivo and a small amount of expression, it could detect the expression of NO in the injured spinal cord only when we used highly sensitive procedure for quantitative measurement of NO by a modified spin-trapping method [23]. To confirm the signal was that of NO, we investigated the effect of an NOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), a strong selective inhibitor of brain and endothelial cell NO synthesis, on the NO spectrum. The spectrum was not seen after intravenous injection of l-NAME (30 mg/kg) 15 min before SCI. After spinal cord compression, the NO level was increased 3.3-fold in the injured region and 4.9-fold in the adjacent central region.

The value of the thiobarbituric acid reactive substances (TBARS) representing the amount of lipid peroxide was 33.1 ± 6.1 nmol/g wet weight in the normal spinal cord, and the value increased after 30 min to 2.2-fold (66.9 ± 5.9) in the injured region and to 1.8-fold (58.5 ± 9.9) in the adjacent central region. Intravenous infusion of l-NAME (30 mg/kg) for 15 min before injury increased the TBARS value in the injured spinal cord to 2.6-fold (86.3 ± 8.1) at 30 min after injury. These results showed the close correlation of NO production and the oxidative stress after mechanical trauma to the spinal cord.


18.3.2 Expression of Constitutive and Inducible NOS in the Spinal Cord


There are several forms of NOS, and we measured the expression of mRNAs of constitutive NOS (c-NOS) and inducible NOS (i-NOS) since c-NOS has been reported to be short-acting and to play protective roles, whereas i-NOS to be long-acting and cytotoxic in the central nervous system. We measured c-NOS mRNA expression before and in the subacute phase after SCI and found that the level of the c-NOS signal did not change after SCI. No appreciable i-NOS signal was detected in RNA from the control spinal cord; it was observed in RNA from 3 h after injury (30-g weight, 15 min compression) and reached maximum after 24 h (Fig. 18.4).

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Fig. 18.4
Change with time in i-NOS mRNA/G3PDH mRNA ratio after compression injury with a 30-g weight for 15 min. The relative amount of i-NOS mRNA was quantified by densitometry and expressed as i-NOS/G3PDH ratio. Points and bars show the mean ± SD of values obtained in three rats at each determination point [18]


18.3.3 Effect of NOS Inhibitor on Lipid Peroxidation and Motor Disturbance After Mechanical Trauma


We investigated the effects of l-NAME on the level of TBARS, MPO activity, and motor function in order to determine the correlation between the level of NO and oxidative injury after injury.

We injected a single dose of l-NAME at 30 mg/kg 15 min before injury intravenously to decrease the activity of c-NOS which is present in the spinal cord at the time of injury. The MPO activity was 27.3 ± 6.6 unit/g net weight in the normal spinal cord and increased to 205.8 ± 18.2 in the injured region 4 h after SCI. Pretreatment with l-NAME increased the MPO activity to 497.2 ± 85.2 in the injured spinal cord 4 h after SCI, and the recovery of motor function was poorer.

Low-dose l-NAME injections (0.1 mg/kg, 4 injections) after injury were used to elucidate the roles of i-NOS induced after injury. Low-dose intravenous injection of l-NAME at this dose 5 min and 3, 6, and 24 h after injury significantly reduced motor disturbance except half a day after injury. Injection of the same dose of l-NAME 6, 24, 48, and 72 h after SCI also significantly reduced motor disturbance (Fig. 18.5).

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Fig. 18.5
Effect of l-NAME treatment on the hind limb motor function after injury to the spinal cord for 5 min with a 30-g weight. The hind limb motor function was assessed every day until 14 days after SCI by Tarlov’s criteria. l-NAME (0.1 mg/kg) was injected into a tail vein 5 min and 3, 6, and 24 h (dot line) or 6, 24, 48, and 72 h (line) after injury. Values are means for ten rats. Values for l-NAME-treated groups are significantly different from those for the control group by the Mann–Whitney U-test at all times except 0.5 day after injury [18]


18.4 Preventive Effects of Lecithinized Superoxide Dismutase (PC-SOD) Against Oxidative Stress [24, 25]


Superoxide dismutase (SOD), an enzyme that scavenges superoxide anions [26], was effective on experimental ischemic SCI [27]. However, its short half-life in vivo and low cell membrane or tissue affinity prevented further development. Lecithinization of SOD (PC-SOD) may increase its half-life and its affinity, which made its applicability higher (Fig. 18.6) [28]. We compared the effects with MP which has been shown to be effective to improve spinal cord function after injury in the experimental models.

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Fig. 18.6
Structure of PC-SOD [24]


18.4.1 Effect of PC-SOD Injection on Lipid Peroxidation in theSpinal Cord Injury


SOD activity in spinal cord tissue increased by 32.4 % (p < 0.01) 4 h after SCI. Intravenous injection of unmodified SOD 30 min after injury did not alter SOD level, but that of PC-SOD significantly increased it by 58.7 % (p < 0.01). These changes were not observed in the plasma [24].

We also examined their effects on lipid peroxidation. The value of TBARS increased from 66.2 ± 12.8 nmol MDA/g tissue in the normal spinal cord to 192.2 ± 16.8 nmol MDA/tissue 1 h after SCI in the injured region. Intravenous bolus infusion of 30 min after SCI reduced the increase in the TBARS value in the injured spinal cord to 142.9 ± 8.2 in the PC-SOD (40,000 units/kg) group, 143.2 ± 5.70 in the MP (30 mg/kg) group, and 83.2 ± 10.6 in the PC-SOD+MP group. PC-SOD and MP had additive effects on the TBARS level [25].


18.4.2 Comparison of the Effect of PC-SOD on Motor Function [25]


Recovery of hind limb motor function after moderate incomplete injury was compared among rats treated with intravenous injection of PC-SOD (40,000 units/kg), that with MP and that with both PC-SOD and MP. The BBB open field locomotor scores of three groups were significantly higher than that of the control group in the first 3 days (p < 0.05). The score of the PC-SOD group increased, whereas that of the MP group showed a temporary decrease from day 3 to 5 and then it gradually recovered. The scores in all groups reached a plateau about 18 days after SCI. The PC-SOD + MP group showed similar tendency to that of the MP group (Fig. 18.7). The angle of rotation in footprint analysis 6 weeks after injury showed significantly better function in PC-SOD group and PC-POD + MP group than MP group (Fig. 18.8).
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Mar 11, 2017 | Posted by in NEUROSURGERY | Comments Off on Oxidative Stress as Secondary Injury Mechanism After Mechanical Trauma in the Spinal Cord

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