In order to evaluate the potential of the catalytic antioxidant MnTBAP for the treatment of SCI, the present study characterized its abilities to scavenge ROS, to protect against oxidative stress in neurons and to ameliorate neurological dysfunction following SCI. The abilities to remove ROS and to improve functional recovery were compared between MnTBAP and MP, in addition to our previous comparison of the antioxidative and neuron protective effects between them  given that MP is the only drug used clinically for treating SCI.
By microdialysis or microcannula sampling and analysis of microdialysates or perfusates by HPLC or spectrophotometrically, we demonstrated that both H2O2 and O2
•- levels in the extracellular space of the rat spinal cord significantly increased following SCI (25 g.cm). The approximately 16% increase in average post-SCI H2O2 and the approximately 60% elevation of the average post-SCI O2
•─ level compared with the pre-injury level are less than the 30% and 100% increases of H2O2 and O2
•- levels induced by a more severe injury (75 g.cm), as we reported previously [16, 17]. Therefore, the production of H2O2 and that of O2
•- are positively significantly correlated with the severity of impact injury.
The abilities of MnTBAP and MP to reduce ROS were compared in vivo and in vitro. We demonstrated for the first time in vivo that the optimal dose of MnTBAP administered directly into the intrathecal space of the cord significantly reduced the elevation of H2O2 in the extracellular space to the basal level (Figure 1) and significantly reduced the elevation of O2
•- by approximately 50% following SCI, compared with the levels in the vehicle-treated animals (Figure 2). In contrast, the standard regimen of MPSS failed to significantly reduce the elevation of O2
•─ (Figure 2). In vitro experiments were carried out to explore the possibly different mechanisms by which MnTBAP and MP attenuate concentrations of H2O2 and O2
•- (Figure 3). We demonstrated that treatment by MnTBAP significantly reduced H2O2 levels to approximately 40% of the levels in ACSF-treated samples, and significantly reduced O2
•- levels to approximately 80% of the levels in ACSF group. In contrast, MP had no effect on levels of either H2O2 or O2
•- compared with those of corresponding ACSF-treated groups. Our in vitro experiment demonstrated that MnTBAP can scavenge H2O2 and O2
•-, whereas MP could not scavenge these ROS. Therefore the ROS-scavenging ability of MnTBAP should contribute to the reduction of SCI-elevated H2O2 and O2
The catalase activity of most metalloporphyrins is less than 1% of that of native catalase . The SOD activity of MnTBAP is 41 units/mg (the unit is defined as the amount of compound that inhibits the reduction of cytochrome c by 50%), and the catalase activity of MnTBAP is only 0.81/min (pseudo first-order rate constant for H2O2 decay) . Our results demonstrated that with its “poor” catalase activity and greater SOD activity, MnTBAP was able to completely remove the overproduced H2O2 but only removed 50% of the overproduced O2
in vivo in our SCI model. Given the optimal dose of MnTBAP (4 mg/kg), approximately 1 mg of MnTBAP was administered to a 250 g rat. Therefore, MnTBAP equivalent to 41 units of SOD activity was administered to a rat. According to this definition, if one unit inhibits the reduction of cytochrome c by 50%, then, 41 units should be much more than enough to inhibit all reduction of cytochrome c by O2
•-. However, in our study 41 units inhibited the reduction of cytochrome c by only 50%, a forty fold lower SOD activity in our in vivo experiment than expected . In contrast, the lower catalase activity of MnTBAP, as reported , brought the SCI-induced H2O2 elevation down to the basal level. Our in vitro experiment, consistent with our in vivo finding, also showed that MnTBAP proved more effective at scavenging H2O2 than it did O2
•- . Although the mechanism of the dramatic difference between the report  and both our in vivo and in vitro findings was not explored in this study, our results support the statement that “MnTBAP is not an SOD mimic, as it has negligible SOD-like activity” by Batinić-Haberle et al. .
The effectiveness of MnTBAP protection against oxidative stress was examined spatially around the injury epicenter. Because the neurons in the cord near the epicenter were killed immediately by acute mechanical injury, they could not be rescued by pharmaceutical intervention. Conversely, the cells far from the epicenter largely remained intact, so treatment should also not have much effect in this area, as reported . Because substantial effects of the drug treatment can only be seen in a limited area, a spatial profile more accurately indicates the area of protection at the cellular level. Our spatial profiles indicated that 4 mg/kg of MnTBAP significantly reduced the numbers of HNE-positive neurons in the sections 1.55 to 2.55 mm rostral to the epicenter (Figure 4) and Ntyr-positive neurons in the sections 1.1 to 3.1 mm rostral to the epicenter (Figure 5) compared with numbers in the vehicle-treated group. MnTBAP at 4 mg/kg reduced MLP and protein nitration in a larger area than in our previous study  in which 1 mg/kg MnTBAP was also given intrathecally: 1 mm in length at 4 mg/kg and 0.5 mm long at 1 mg/kg for MLP, and 2.0 mm in length at 4 mg/kg and 0.5 mm at 1 mg/kg for protein nitration. The area of significant protection also started approximately 1 mm closer to the epicenter for 4 mg/kg than for 1 mg/kg. This comparison indicated that protective effects of MnTBAP are correlated with doses administered. The results in the present study together with our previous publications [44, 45, 53] indicated that MnTBAP ameliorates secondary damage within the boundary area. SCI treatment should be greatly improved by combining antioxidant therapy to save cells in the boundary area with other manipulations such as transplantation of stem cells, Schwann cells, and nanoparticles, etc. to restore the neurons and to repair and regeneration of axons in the injury epicenter. [59–61].
RS can cause injury by directly oxidizing the major cellular components. Sulfhydryl group in proteins is very susceptible to oxidation, nitration, nitrosation, and nitrosylation. Since the oxidative defense enzymes, such as SODs, catalase, GPx, thioredoxin, etc., are all sulfhydryl-containing enzymes, they may suffer function-distorting oxidative modification. MnTBAP scavenges SCI-induced RS, thereby protecting against oxidation and nitration of these antioxidant enzymes – helping restoration of their enzymatic activity. Functional recovery of defense enzymes, in turn, further reduces RS production. This positive feedback mechanism enhances the effects of MnTBAP protection. RS can cause injury by acting as intracellular death signals that lead to changes in expression of proteins, and by acting as modulators of the redox state. RS also have physiological functions. In redox cycles, the primary signal transducers are not large proteins but small redox-active molecules. ROS (e.g. O2
•─, H2O2), and RNS (e.g. nitric oxide, S-nitrosothiols and ONOO-) can mediate redox signaling . Therefore, in addition to catalytically scavenging a wide range of RS, other mechanisms such as modulating RS-based redox signaling pathways, and regulating cellular transcription activity may also contribute to the beneficial effects of metalloporphyrins . It was reported that MnTBAP attenuated the nuclear translocation of apoptosis-inducing factor and the subsequent DNA fragmentation induced by ROS after permanent focal cerebral ischemia in mice . So, in addition to their enzymatic catalyzing scavenging activities, more complicated mechanisms may be involved in our in vivo finding that MnTBAP reduced elevation of deleterious ROS, attenuated oxidative stress in neurons, and improved functional recovery after SCI.
The mechanisms by which the steroid MP protect against MLP and neurological deficits lies in its anti-oxidative and anti-inflammatory activities. In contrast to the effects of MnTBAP, our in vitro results demonstrated that MP had no effect on the levels of H2O2 and O2
•-. This is consistent with the published report that MP does not directly trap radicals . The activity of the oxidative defense enzyme GPx in erythrocytes of the rat was inhibited after SCI and MP restored GPx activity to near-basal levels . Similar tendencies were also found for the activities of catalase and GPx in the spinal cord tissues of rats following SCI and MP treatment . This suggests that the in vivo reduction of H2O2 by MP following SCI probably occurs indirectly through pathways, such as anti-inflammatory activities  and restoration of the activities of oxidative defense enzymes [65, 66]. Our previous study demonstrated that MP significantly reduced the extracellular release of prostaglandin F2α (PGF2α), a membrane hydrolysis product, following SCI. It blocked PGF2α-induced •OH and MDA production. However, unlike MnTBAP, MP did not significantly reduce Fenton’s reagent-induced MDA production in the rat spinal cord, even though the MDA concentration was below the concentration induced by PGF2α. This suggests that 1) MP cannot directly scavenge •OH, 2) its “antioxidant” effect is probably a consequence of interrupting the production of toxic hydrolysis products, and 3) the MLP is triggered by hydrolysis product-induced free radicals . Therefore, the broad-spectrum RS scavenger MnTBAP better attenuates oxidative damage than does MP.
We previously measured the time course of protein nitration by counting the Ntyr-positive cells in sections stained by the anti-Ntyr antibody at different times post-SCI . We found that protein nitration peaked at 12 to 24 h post-SCI. In a recently published study, Carrico et al. reported that Ntyr-positive and HNE-positive staining increased starting from 3 h to as long as 1 and 2 weeks post-SCI . Christie et al. reported that after acute SCI the levels of MDA rose quickly at 4 h, returned to baseline at 12 h, and increased again by 24 h post-SCI, and the elevation was sustained for 120 h post-SCI . Luo et al. reported that HNE-protein adducts increased in the damaged cord as early as 4 h after SCI, reached a peak level at 24 h, and remained significantly elevated up to 7 days after SCI . Therefore, in the present study, the spatial profiles of MnTBAP protection against oxidative stress were measured at 24 h post-SCI. This time point was within the peak region of protein nitration, and production of MDA and HNE-protein adducts.
Although the regimen of MPSS applied in our study was the standard one for the clinical treatment of SCI, it was originally developed by using experimental animals. This regimen was reported as the optimal dose in cats compared with 15 and 60 mg/kg . The bolus dose of 30 mg/kg plus follow-up administration was also used in experimental SCI of rats [71–73]. In a dose–response study using a ventral compression injury model, the best results were yielded when MPSS was administered in the rats with a bolus dose of 30 mg/kg at 30 min post-SCI followed by a second injection of 15 mg/kg (a total of 45 mg/kg) or with a 60 mg/kg bolus at 30 min post-SCI . This demonstrates that the standard regimen of MPSS for the clinical treatment of SCI is also suitable for rats. Therefore, the standard clinical regimen of MPSS was applied in the present study.
Administering MP within 8 hours improves neurological recovery in SCI. However, the high dose of MP is ineffective and possibly even deleterious when given more than 8 hours post-SCI [43, 75, 76]. This indicates that 8 h is an effective time window for MP treatment. In many studies of experimental SCI treated with MP, MPSS was given immediately following SCI (0 h post-SCI). Unfortunately, in clinical practice patients can rarely be treated at 0 h post-injury. We demonstrated that the time window for MnTBAP to reduce oxidative damage and neuron death was at least 12 h after SCI . Therefore, in addition to comparing the efficacy of 4 h post-SCI treatment with MnTBAP and MP to protect against oxidative stress and cell death as we reported , the present study further compared the effectiveness of 4 h post-SCI treatment with MnTBAP and MP in functional recovery after SCI. This is not only within the effective time window for both MP and MnTBAP, but also a more practical time window for clinical usage. We demonstrated that post-SCI treatment with 10 mg/kg MnTBAP (ip), or with the standard MP regimen (iv), both significantly increased BBB and inclined plane scores compared to vehicle treatment. However, the scores in MnTBAP-treated animals were significantly better than in MP-treated animals, demonstrating that post-SCI treatment with MnTBAP is more effective than MP for improving neurological dysfunction after SCI.
MnTBAP was reported as poorly penetrating the blood brain barrier , so it is not a favorable candidate for antioxidant therapy for central nervous system injury. We also demonstrated that the MP’s blood–spinal cord barrier penetrating ratio was much higher than MnTBAP (20% versus 4%) . However, our results of behavioral tests demonstrated that with lower ability of penetrating the blood–spinal cord barrier, MnTBAP reached appropriate targets and more effectively improved the functional recovery than the standard treatment of SCI by MP, further supporting the candidacy of MnTBAP in treating SCI.