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Traumatic spinal cord injury (SCI) enhances the activity of S-nitrosoglutathione reductase (GSNOR) and inhibits the mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity, resulting in prolonged and sustained pain and functional deficits. This study's objective was to test the hypotheses that GSNOR's specific inhibitor N6022 mitigates pain and improves functional recovery in a mouse model of SCI. Furthermore, the degree of recovery is enhanced and the rate of recovery is accelerated by an ALDH2 activator Alda-1. Using both wild-type and GSNOR - / - mice, the SCI model deployed for groups was contusion at the T9-T10 vertebral level. The enzymatic activity of GSNOR and ALDH2 was measured, and the expression of GSNOR and ALDH2 was determined by western blot analysis. Functional improvements in experimental animals were assessed with locomotor, sensorimotor, and pain-like behavior tests. Wild-type SCI animals had enhanced GSNOR activity and decreased ALDH2 activity, leading to neurovascular dysfunction, edema, and worsened functional outcomes, including locomotor deficits and pain. Compared to wild-type SCI mice, GSNOR - / - mice had better functional outcomes. Monotherapy with either GSNOR inhibition by N6022 or enhanced ALDH2 activity by Alda-1 correlated well with functional recovery and lessened pain. However, combination therapy provided synergistic pain-relieving effects and more significant functional recovery compared with monotherapy. Conclusively, dysregulations in GSNOR and ALDH2 are among the causative mechanisms of SCI injury. Either inhibiting GSNOR or activating ALDH2 ameliorates SCI. Combining the specific inhibitor of GSNOR (N6022) with the selective activator of ALDH2 (Alda-1) provides greater protection to the neurovascular unit and confers greater functional recovery. The study is novel, and the combination therapy (N6022 + Alda-1) possesses translational potential., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The glycinamide ribonucleotide transformylase (GART) gene, a trifunctional polypeptide, has phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase activity, and is required for de novo purine biosynthesis. GART is highly conserved in vertebrates. Alternative splicing of GART results in two transcript variants encoding different isoforms. However, the expression and function of GART in the central nervous system lesion are still unclear. In this study, we used a traumatic spinal cord injury (SCI) model in adult Sprague-Dawley rats and investigated the dynamic changes of GART protein expression in the spinal cord. Western blot analysis revealed that GART was present in sham-operated spinal cord. It gradually increased, reached a peak at day 3 after SCI, and then declined during the following days. Double immunofluorescence staining revealed a widespread of GART, and the majority of GARTs are detected in astrocytes. After injury, GART expression was increased predominantly in astrocytes, positively correlated with the highly expressed proliferating cell nuclear antigen (PCNA). Knockdown of GART expression in cultured primary astrocytes by siRNA revealed that expression of GART in astrocytes plays a role in the LPS-induced release of pro-inflammatory factors, such as TNF-α and IL-6. These results showed that GART may participate in the pathophysiology of SCI, and more research is needed to have a good understanding of its function and mechanism., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Histone deacetylases (HDAC) inhibitors including valproic acid (VPA) have emerged as a promising therapeutic intervention in neurological disorders. We investigated the levels of acetylated histone and the therapeutic potential of VPA in a rat model of spinal cord injury (SCI). At different time points (12 h, 1 day, 3 days, 1 week and 2 weeks) after SCI or sham surgery, the spinal cords were collected to evaluate the levels of acetylated histone H3 (Ac-H3) and H4 (Ac-H4). VPA or vehicle was injected for 1 week starting immediately after SCI and histone acetylation, apoptosis, as well as neurobehavior were observed to test the effect of VPA. The levels of Ac-H3 and Ac-H4 in the injured spinal cord started to significantly decrease as early as day 1, and remained below those in uninjured controls for at least 2 weeks after SCI. Injection of VPA markedly prevented the reductions of Ac-H3 and Ac-H4, upregulated the expressions of Hsp70 and Bcl-2, reduced apoptosis and finally promoted locomotion recovery. Our data demonstrated that SCI led to marked reduction in histone acetylation; VPA was neuroprotective in the SCI model, and the mechanism may involve HDAC inhibition and protective proteins induction., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Primary trauma to the spinal cord triggers a cascade of cellular and molecular events that promote continued tissue damage and expansion of the lesion for extended periods following the initial injury. Oxidative and nitrosative stresses play an important role in progression of spinal cord injury (SCI). In an attempt to explore the biochemical origin of oxidative/nitrosative stress associated with secondary SCI, we studied expression of the superoxide (O2*-)-generating enzyme, NAD(P)H oxidase, antioxidant enzymes [superoxide dismutase (CuZn SOD, Mn SOD), catalase, glutathione peroxidase (GPX)], nitric oxide synthases (NOS) and a byproduct of NO-O2*- interaction (nitrotyrosine) in the spinal cord tissues of rats 16 h and 14 days after surgical resections of a 5-mm segment of the cord below T8 or sham-operation. Immunodetectable NAD(P)H oxidase subunits (gp91phox and P67phox), Mn SOD, inducible NOS (iNOS), endothelial NOS (eNOS), and nitrotyrosine were elevated in the transected cords on day 1 and day 14. Neuronal NOS (nNOS) was unchanged on day 1 and significantly depressed on day 14. GPX was unchanged on day 1 and significantly elevated on day 14. Catalase was unchanged in the cord tissue surrounding the transection site at both points. Thus, concurrent upregulations of NAD(P)H oxidase, eNOS and iNOS (but not nNOS), work in concert to maintain oxidative and nitrosative stress in the injured cord tissue.

Glucocorticoids (GC) provide neuroprotection and early recovery after spinal cord injury (SCI). While several mechanisms were proposed to account for these effects, limited information exists regarding GC actions in sensory areas of the spinal cord. Presently, we studied the time course of Fos expression, and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical staining to monitor neuronal responses to SCI with or without GC treatment. Rats with sham-operation or transection at the thoracic level (T7-T8) received vehicle or 5 mg/kg of the GC dexamethasone (DEX) at 5 min post-lesion and were sacrificed 2 or 4 h after surgery. Another group of SCI rats received vehicle or intensive DEX treatment (5 min, 6 h, 18 h and 46 h post-lesion) and were sacrificed 48 h after surgery. The number of NADPH-d positive neurons or Fos immunoreactive nuclei was studied by computer-assisted image analysis in superficial dorsal horn (Laminae I-III) and central canal area (Lamina X) below the lesion. While constitutive Fos immunoreactive nuclei were sparse in controls, SCI increased Fos expression at 2 and 4 h after injury. DEX treatment significantly enhanced the number of Fos positive nuclei in Laminae I-III by 4 h after transection, although the response was not maintained by intensive steroid treatment when tested at 48 h after SCI. NADPH-d positive neurons in Laminae I-III increased at 2 and 4 h after SCI while a delayed increased was found in central canal area (Lamina X). DEX treatment decreased NADPH-d positive neurons to sham-operated levels at all time points examined. Thus, while GC stimulation of Fos suggests activation of neurons involved in sympathetic outflow and/or pain, down-regulation of NADPH-d indicates attenuation of nociceptive outflow, considering the role of enzyme-derived nitric oxide in pain-related mechanisms. Differential hormonal effects on these molecules agree with their localization in different cell populations.

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy., (Copyright 1998 Elsevier Science B.V.)

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Light microscopic enzyme histochemistry was employed to study the alterations of acetylcholinesterase (AChE) within lumbosacral ventral horn neurons at survival times of 1, 4, 7, 14, 28, 60, and 90 days after low thoracic spinal cord hemisection in adult rats. The intensity of histochemical staining was quantified using densitometric techniques. Virtually all ventral horn neurons of sham-operated and unoperated animals, which served as controls, displayed intense AChE staining. Hemisection of the spinal cord induced a transient ipsilateral decrease of AChE staining in most neuronal cell bodies and in the neuropil of lamina IX at all segmental levels caudal to the lesion. Quantitative analysis of representative segments revealed a reduction of AChE in the ventral horn during a postoperative (p.o.) period of 1 to 28 days followed by a phase of recovery over the next two months. AChE activity still remained slightly reduced, even at 90 days p.o. The transient decrease in AChE is a well-known metabolic response of axotomized motoneurons. However, the observed changes of AChE reactivity in intact motoneurons ipsilateral and caudal to the hemisection are presumably induced by the interruption of supraspinal descending pathways. These metabolic changes may functionally affect the whole motor unit and be involved in the disturbances of motor function following spinal cord injury.

BW755C is an inhibitor of both cyclo-oxygenase and lipoxygenase, which has been found to have protective effects after myocardial ischemia in dogs. Impact injury to the spinal cord is associated with tissue ischemia as well as with the accumulation of eicosanoids. In the present studies we evaluated the effects of BW755C after traumatic spinal cord injury in rats. Drug treatment reduced thromboxane B2 levels and improved neurological recovery as compared to treatment with equal-volume physiological saline. The findings suggest that this drug or related compounds may be useful for the treatment of clinical spinal cord injury.