Valproic acid improves locomotion in vivo after SCI and axonal growth of neurons in vitro

Previous studies have found that valproic acid (VPA), a histone deacetylases (HDAC) inhibitor, improves outcomes in a rat model of spinal cord injury (SCI). The study here aimed to further illuminate the neuroprotective effects of VPA against SCI, both in vivo and in vitro. First, spinal cord injury was performed in rats using NYU impactor. Delayed VPA injection (8 h following SCI) significantly accelerated locomotor recovery. VPA therapy also suppressed SCI-induced hypoacetylation of histone and promoted expressions of BDNF and GDNF. Next, the influence of VPA on axonal growth inhibited by a myelin protein was tested. Neurons from embryonic spinal cord or hippocampus were cultured on plates coated with Nogo-A peptide, and escalating concentrations of VPA were added into the cultures. VPA treatment, in a concentration dependent manner, allowed neurons to overcome Nogo-A inhibition of neurite outgrowth. Meanwhile, VPA exposure increased the level of histone acetylation and expression of BDNF in spinal neurons. Cumulatively, these findings indicate that VPA is possibly a promising medication and deserves translational trials for spinal cord injury.     

雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究

目的探讨雷公藤甲素（TP）与甲泼尼龙（MP）调节细胞自噬和凋亡促进脊髓损伤（SCI）修复的作用机制,为临床替代MP治疗SCI提供理论依据和新的可选择性药物。 方法选取60只雌性Thy-YFP转基因小鼠建立SCI模型,按照随机数字表法分为假手术组（Sham组）、DMSO溶液处理组（DMSO组）、MP实验组和TP实验组,每组15只。TP实验组、DMSO组及Sham组小鼠分别于术后立即腹腔注射TP（0.002 mg/10 g）、等量5%DMSO、0.9%NaCl溶液,连续给药7 d;MP实验组小鼠于术后30 min、6 h、24 h腹腔注射MP溶液（0.3 mg/10 g）。采用BMC评价运动功能,HE染色及Nissl染色法检测脊髓组织学变化,采用免疫印迹及免疫荧光染色检测自噬相关蛋白（Beclin-1、LC3B、p62）及凋亡相关蛋白（Bcl-2、Bax、caspase-3）的水平。 结果TP实验组干预后SCI小鼠BMS运动功能评分随着时间推移逐渐升高,神经元数量增多,自噬相关蛋白Beclin-1、LC3B上调且p62降低,细胞凋亡相关蛋白caspase-3、Bax降低且抗凋亡蛋白Bcl-2升高;无论是运动功能评价、组织学变化,还是SCI后细胞自噬增强、细胞凋亡减少,TP实验组均优于DMSO组（P<0.05）,但与MP实验组比较差异均无统计学意义（P>0.05）。 结论TP在急性SCI中通过上调自噬、抑制凋亡可以促进损伤后的脊髓运动功能恢复,与MP相比对SCI具有相同的潜在保护作用。     

The neuroprotective effect of treatment of valproic Acid in acute spinal cord injury

Objective

Valproic acid (VPA), as known as histone deacetylase inhibitor, has neuroprotective effects. This study investigated the histological changes and functional recovery from spinal cord injury (SCI) associated with VPA treatment in a rat model.

Methods

Locomotor function was assessed according to the Basso-Beattie-Bresnahan scale for 2 weeks in rats after receiving twice daily intraperitoneal injections of 200 mg/kg VPA or the equivalent volume of normal saline for 7 days following SCI. The injured spinal cord was then examined histologically, including quantification of cavitation.

Results

Basso-Beattie-Bresnahan scale scores in rats receiving VPA were significantly higher than in the saline group (p<0.05). The cavity volume in the VPA group was significantly reduced compared with the control (saline-injected) group (p<0.05). The level of histone acetylation recovered in the VPA group, while it was significantly decreased in the control rats (p<0.05). The macrophage level was significantly decreased in the VPA group (p<0.05).

Conclusion

VPA influences the restoration of hyperacetylation and reduction of the inflammatory reaction resulting from SCI, and is effective for histology and motor function recovery.     

Rapamycin increases neuronal survival,reduces inflammation and astrocyte proliferation after spinal cord injury

Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI.In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.     

Lithium promotes recovery of neurological function after spinal cord injury by inducing autophagy

Lithium promotes autophagy and has a neuroprotective effect on spinal cord injury(SCI); however, the underlying mechanisms remain unclear. Therefore, in this study, we investigated the effects of lithium and the autophagy inhibitor 3-methyladenine(3-MA) in a rat model of SCI. The rats were randomly assigned to the SCI, lithium, 3-MA and sham groups. In the 3-MA group, rats were intraperitoneally injected with 3-MA(3 mg/kg) 2 hours before SCI. In the lithium and 3-MA groups, rats were intraperitoneally injected with lithium(LiCl; 30 mg/kg) 6 hours after SCI and thereafter once daily until sacrifice. At 2, 3 and 4 weeks after SCI, neurological function and diffusion tensor imaging indicators were remarkably improved in the lithium group compared with the SCI and 3-MA groups. The Basso, Beattie and Bresnahan locomotor rating scale score and fractional anisotropy values were increased, and the apparent diffusion coefficient value was decreased. Immunohistochemical staining showed that immunoreactivities for Beclin-1 and light-chain 3 B peaked 1 day after SCI in the lithium and SCI groups. Immunoreactivities for Beclin-1 and light-chain 3 B were weaker in the 3-MA group than in the SCI group, indicating that 3-MA inhibits lithium-induced autophagy. Furthermore, NeuN+ neurons were more numerous in the lithium group than in the SCI and 3-MA groups, with the fewest in the latter. Our findings show that lithium reduces neuronal damage after acute SCI and promotes neurological recovery by inducing autophagy. The neuroprotective mechanism of action may not be entirely dependent on the enhancement of autophagy, and furthermore, 3-MA might not completely inhibit all autophagy pathways.     

Inhibition of the Ca2?-dependent K? channel, KCNN4/KCa3.1, improves tissue protection and locomotor recovery after spinal cord injury

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K? channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca2? signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.     

Sodium valproate at therapeutic concentrations changes Ca2+ response accompanied with its weak inhibition of protein kinase C in human astrocytoma cells

Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of gamma-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 microM) on the elevation of intracellular free calcium concentration ([Ca(2+)](i)) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 microM VPA for 2 min did not change the carbachol-induced [Ca(2+)](i) elevation. Treatment with 300 and 600 microM VPA for 48 h, however, reduced the elevation. Since we have shown that Li(+) reduced carbachol-induced [Ca(2+)](i) elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca(2+)response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17-22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 microM VPA for 24 or 48 h slightly increased the B(max) value, but not the K(d) value, in the binding of [(3)H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca(2+)](i) elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca(2+)](i) elevation may play a role in the treatment of mood disorder.     

A monoclonal antibody to CD11d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment

The accumulation of inflammatory cells in the lesion of a spinal cord injury (SCI) enhances secondary damage, resulting in further neurological impairment. High-dose methylprednisolone (MP) treatment is the only accepted treatment for inflammation secondary to human SCI but is minimally effective. Using a rat SCI model, we devised an anti-inflammatory treatment to block the infiltration of neutrophils and hematogenous monocyte/macrophages over the first 2 days postinjury by targeting the CD11dCD18 integrin. Anti-CD11d mAb administration following SCI effectively reduced neutrophil and macrophage infiltrate into lesions by 70% and 36%, respectively, over the first 72 h post-SCI. MP also reduced neutrophil and macrophage infiltrate by 60% and 28%, respectively, but by different mechanisms. The immunosuppression caused by anti-CD11d treatment was not sustained, as inflammatory cell numbers were not different from those observed in untreated SCI control animals at 7 days postinjury. In contrast, in MP-treated animals, the number of macrophages was still suppressed in the lesion while neutrophil numbers were significantly increased. These results suggest that anti-CD11d mAb treatment following SCI will minimize the destructive actions associated with early, uncontrolled leukocyte infiltration into the lesion while permitting the positive wound healing effects of macrophages at later time points.     

The systemic inflammatory response after spinal cord injury damages lungs and kidneys

Spinal cord injury (SCI) triggers a well characterized, acute, local inflammation leading to secondary damage at the lesion site. Another little recognized problem may be the activation of circulating inflammatory cells that potentially damage tissues outside the cord. We investigated this problem using severe clip-compression SCI in rats. We studied systemic inflammation after SCI and its effects on lungs and kidneys, as dysfunction of these organs is a frequent, early complication after SCI. From 2-24 h after SCI, the number of circulating neutrophils (especially immature cells) significantly increased by 3-10 fold. Flow cytometry experiments revealed that SCI transiently activates these neutrophils, causing increased oxidative responses to phorbolmyristic acid at 2 h after SCI; then, from 4-24 h, the neutrophils were less responsive. Neutrophil longevity was increased (30-50% decrease in apoptosis) at 2-8 h after SCI. Immunohistochemical analyses demonstrated the invasion of neutrophils into lungs and kidneys (2 h-7 d after SCI) and more phagocytic macrophages in lungs (12 h, 3 d after SCI). Myeloperoxidase and matrix metalloproteinase-9 activity in lung and kidney homogenates increased (12 h-7 d after SCI). Expression of COX-2 increased and lipid peroxidation also occurred within this time. Control experiments inducing local cord damage by excitotoxic quisqualate injection verified that SCI per se is sufficient to trigger systemic inflammation and organ damage. In summary, SCI mobilizes and activates neutrophils that then migrate into visceral organs, a phenomenon occurring in parallel with their well-known entry into the cord injury site. The systemic inflammatory response to SCI should be targeted in the development of new therapeutic strategies to treat SCI.     

Glucocorticoid effects on Fos immunoreactivity and NADPH-diaphorase histochemical staining following spinal cord injury

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.     

Evidence for the role of PI(3) -kinase-AKT-eNOS signalling pathway in secondary inflammatory process after spinal cord compression injury in mice

Endothelial nitric oxide synthase (eNOS) is a dynamic enzyme tightly controlled by co‐ and post‐translational lipid modifications, phosphorylation and regulated by protein–protein interactions. In this study we have pharmacologically modulated the activation of eNOS, at different post‐translational levels, to assess the role of eNOS‐derived NO and regulatory mechanisms in tissue damage associated with spinal cord injury (SCI). SC trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four‐level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, and production of inflammatory mediators, tissue damage and apoptosis. LY294002, an inhibitor of phosphatidylinositol 3‐kinase that initiates Akt‐catalysed phosphorylation of eNOS on Ser1179, was administered 1 h before the induction of SCI; 24 h after SCI sections were taken for histological examination and for biochemical studies. In this study we clearly demonstrated that pre‐treatment with LY294002 reversed the increased activation of eNOS and Akt observed following SCI, and developed a severe trauma characterized by oedema, tissue damage and apoptosis (measured by TUNEL staining, Bax, Bcl‐2 and Fas‐L expression). Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity. Overall these results suggest that activation of the Akt pathway in SC tissue subject to SCI is a protective event, triggered in order to protect the injured tissue through a fine tuning of the endothelial NO pathway.     

Valproate-induced thrombocytopenia: a prospective monotherapy study

PURPOSE: The frequency of valproate (VPA)-induced thrombocytopenia varied widely in previous studies, due to methodological differences. Our objective was to evaluate the relationship between trough VPA plasma levels and platelet counts and assess risk factors for the development of thrombocytopenia. METHODS: Patients with refractory partial epilepsy were enrolled in this double-blind, multicenter, concentration-response trial that evaluated the efficacy and safety of high versus low trough plasma VPA concentrations following administration of divalproex sodium as monotherapy. Trough VPA concentrations and concomitant platelet counts were drawn at baseline and intermittently throughout the 24-week trial. Bivariate correlations and multivariate stepwise regression analysis were performed between platelet counts and multiple variables. A logistic regression analysis was done to determine the probability of developing thrombocytopenia at various VPA levels. RESULTS: A total of 851 VPA levels and concomitant platelet counts were analyzed in 265 patients. Of these, 17.7% of patients experienced at least one episode of thrombocytopenia (platelet count < or = 100,000/microl) after exposure to divalproex sodium. A significant negative correlation was found between VPA levels and platelet counts. Women were significantly more likely to develop thrombocytopenia. The probability of developing thrombocytopenia substantially increased at trough VPA levels above 100 microg/ml in women and above 130 microg/ml in men. DISCUSSION: Our data strongly support a causal relationship between rising plasma VPA levels and reduced platelet counts, with additional risk factors including female gender and lower baseline platelet counts.     

CD11d integrin blockade reduces the systemic inflammatory response syndrome after spinal cord injury

Traumatic injury to the spinal cord triggers a systemic inflammatory response syndrome (SIRS), in which inflammatory cells from the circulation invade organs such as the liver, lung and kidney, leading to damage of these organs. Our previous study (Gris, et al, Exp. Neurol, 2008) demonstrated that spinal cord injury (SCI) activates circulating neutrophils that then invade the lung and kidney from 2 to 24 h after injury, increasing myeloperoxidase activity, cyclooxygenase-2 and matrix metalloproteinase-9 expression and lipid peroxidation in these organs. The present study was designed to ascertain whether a treatment that limits the influx of leukocytes into the injured spinal cord would also be effective in reducing the SIRS after SCI. This treatment is intravenous delivery of a monoclonal antibody (mAb) against the CD11d subunit of the CD11d/CD18 integrin expressed by neutrophils and monocytes. We delivered the anti-CD11d mAb at 2 h post moderate clip compression SCI at the 4th or 12th thoracic segments and assessed inflammation, oxidative activity and cellular damage within the lung, kidney and liver at 12 h post-injury. In some analyses we compared high and low thoracic injuries to evaluate the importance of injury level on the intensity of the SIRS. After T4 injury, treatment with the anti-integrin mAb reduced the presence of neutrophils and macrophages in the lung, with associated decreases in expression of NF-κB and oxidative enzymes and in the concentration of free radicals in this organ. The treatment also reduced lipid peroxidation, protein nitration and cell death in the lung. The anti-CD11d treatment also reduced the inflammatory cells within the kidney after T4 injury, as well as the free radical concentration and amount of lipid peroxidation. In the liver, the mAb treatment reduced the influx of neutrophils but most of the other measures examined were unaffected by SCI. The inflammatory responses within the lung and kidney were often greater after T4 than T12 injury. Clinical studies show that SIRS, with its associated organ failure, contributes significantly to the morbidity and mortality of SCI patients. This anti-integrin treatment may block the onset of SIRS after SCI.     

Improved seizure control by alternating therapy of levetiracetam and valproate in epileptic rats

Purpose: Tolerance to drug treatment is a serious problem in the treatment of epilepsy. We previously showed that tolerance to levetiracetam (LEV) developed within 4 days after the start of the treatment in a rat model for spontaneous seizures after electrically induced status epilepticus. In the current study we tested whether the development of tolerance to LEV could be prevented by alternating between LEV and valproate (VPA) treatment. Methods: Before starting the alternating therapy with LEV and VPA (3 day LEV–3 day VPA, two cycles), we assessed the efficacy of VPA monotherapy by administering VPA to chronic epileptic rats via osmotic minipumps during 7 days. The anticonvulsive effects were determined by continuous video‐EEG (electroencephalography) monitoring, and the concentration of VPA and LEV was measured in plasma using gas chromatography. Results: VPA significantly suppressed spontaneous seizures in chronic epileptic rats for 5 days. Hereafter, seizure frequency increased to pretreatment values despite adequate VPA blood levels. Seizure duration was reduced for 6 days during treatment. Seizure severity was reduced throughout the 7‐day treatment period. Alternating treatment of LEV and VPA did not prevent development of tolerance; however, seizures were suppressed significantly longer compared to VPA and LEV monotherapy. Conclusions: Because alternating treatment with LEV and VPA led to a prolonged effective seizure control in the animal model, it would be worthwhile to explore the possibilities of using an alternating treatment protocol in pharmacoresistant patients in whom an effective treatment is hampered by tolerance to antiepileptic drugs.     

PAF antagonist treatment reduces pro-inflammatory cytokine mRNA after spinal cord injury

Platelet-activating factor (PAF) is a pro-inflammatory molecule which contributes to secondary damage after spinal cord injury (SCI).To test if PAF contributes to cytokine induction following SCI, female Long-Evans rats were pretreated with the PAF antagonist WEB 2170 prior to receiving a contusion injury at spinal cord level T10 using the NYU impactor. RNase protection assay (RPA) analysis revealed that IL-1alpha mRNA peaked at I h post-injury while IL-1beta and IL-6 mRNA levels were higher and peaked at 6 h.TNF-alpha mRNA was almost undetectable. All mRNA levels approached baseline by 24 h. Treatment with WEB 2170 (1 mg/kg, i.p.) 15 min prior to injury significantly decreased mRNA levels for all three cytokines at 6 h post-injury, but not at I h post-injury. These results demonstrate a role for PAF in proinflammatory cytokine induction after SCI.     

Valproic Acid Increases Expression of Neuronal Stem/Progenitor Cell in Spinal Cord Injury

Objective

This study investigates the effect of valproic acid (VPA) on expression of neural stem/progenitor cells (NSPCs) in a rat spinal cord injury (SCI) model.

Methods

Adult male rats (n=24) were randomly and blindly allocated into three groups. Laminectomy at T9 was performed in all three groups. In group 1 (sham), only laminectomy was performed. In group 2 (SCI-VPA), the animals received a dose of 200 mg/kg of VPA. In group 3 (SCI-saline), animals received 1.0 mL of the saline vehicle solution. A modified aneurysm clip with a closing force of 30 grams was applied extradurally around the spinal cord at T9, and then rapidly released with cord compression persisting for 2 minutes. The rats were sacrificed and the spinal cord were collected one week after SCI. Immunohistochemistry (IHC) and western blotting sample were obtained from 5 mm rostral region to the lesion and prepared. We analyzed the nestin immunoreactivity from the white matter of ventral cord and the ependyma of central canal. Nestin and SOX2 were used for markers for NSPCs and analyzed by IHC and western blotting, respectively.

Results

Nestin and SOX2 were expressed significantly in the SCI groups but not in the sham group. Comparing SCI groups, nestin and SOX2 expression were much stronger in SCI-VPA group than in SCI-saline group.

Conclusion

Nestin and SOX2 as markers for NSPCs showed increased expression in SCI-VPA group in comparison with SCI-saline group. This result suggests VPA increases expression of spinal NSPCs in SCI.     

Role of autophagy in early brain injury after experimental subarachnoid hemorrhage

Autophagy is a self-degradative process and it plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, and eliminating intracellular pathogens. Previous studies have demonstrated that autophagy pathway was activated in brain after experimental subarachnoid hemorrhage (SAH); however, the role of autophagy in the pathogenesis of early brain injury (EBI) following SAH remains unknown. Experiment 1 aimed to investigate the time–course of the autophagy in the cortex following SAH. In experiment 2, we chose the maximum time pointof autophagy activation and assessed the effects of rapamycin (RAP, autophagy activator) and 3-methyladenine (3-MA, autophagy inhibitor) on regulation of EBI. All SAH animals were subjected to injection of 0.3 ml fresh arterial, nonheparinized blood into prechiasmatic cistern in 20 s. As a result, microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased at the early stage of SAH and their expressions peaked at 24 h after SAH. In RAP-treated group, the early brain damage such as brain edema, blood–brain barrier (BBB) impairment, cortical apoptosis, and clinical behavior scale was significantly ameliorated in comparison with vehicle-treated SAH rats. Conversely, 3-MA decreased expression of LC3 and beclin-1, increased the average value of brain edema and BBB disfunction, and aggravated neurological deficits. Our results suggest that autophagy pathway is activated in the brain after SAH and may play a beneficial role to EBI development.     

Pharmacological evidence for a role of peroxynitrite in the pathophysiology of spinal cord injury

Evidence suggests that the reactive oxygen species peroxynitrite (PN) is an important player in the pathophysiology of acute spinal cord injury (SCI). In the present study, we examined the ability of tempol, a catalytic scavenger of PN-derived free radicals, to alleviate oxidative damage, mitochondrial dysfunction and cytoskeletal degradation following a severe contusion (200 kdyn force) SCI in female Sprague-Dawley rats. PN-mediated oxidative damage in spinal cord tissue, including protein nitration, protein oxidation and lipid peroxidation was significantly reduced by acute tempol treatment (300 mg/kg, i.p. within 5 min post-injury). Injury-induced mitochondrial respiratory dysfunction, measured after 24 h in isolated mitochondria, was partially reversed by tempol along with an attenuation of oxidative damage to mitochondrial proteins. Mitochondrial dysfunction disrupts intracellular Ca²⁺ homeostasis contributing to calpain-mediated axonal cytoskeletal protein (α-spectrin, 280 kD) degradation. Increased levels of α-spectrin breakdown proteins (SBDP 145 kD and 150 kD) were significantly decreased at 24 h in tempol-treated rats indicative of spinal axonal protection. However, a therapeutic window analysis showed that the axonal cytoskeletal protective effects require tempol dosing within the first hour after injury. Nevertheless, these findings are the first to support the concept that PN is an important neuroprotective target in early secondary SCI, and that there is a mechanistic link between PN-mediated oxidative compromise of spinal cord mitochondrial function, loss of intracellular Ca²⁺ homeostasis and calpain-mediated proteolytic axonal damage.     

Potential neuroprotective effect of Anakinra in spinal cord injury in an in vivo experimental animal model

Objective:

To evaluate the therapeutic effects of inhibiting interleukin-1 beta (IL-1β) in vivo using Anakinra in an experimental model of spinal cord injury (SCI).

Methods:

All experimental procedures were performed in the animal laboratory of Ankara Education and Research Hospital, Ankara, Turkey between August 2012 and May 2014. The SCI was induced by applying vascular clips to the dura via a 4-level T5-T8 laminectomy. Fifty-four rats were randomized into the following groups: controls (n = 18), SCI + saline (n = 18), and SCI + Anakinra (n = 18). Spinal cord samples were obtained from animals in both SCI groups at one, 6, and 24 hours after surgery (n = 6 for each time point). Spinal cord tissue and serum were extracted, and the levels of IL-1β, malondialdehyde, glutathione peroxidase, superoxide dismutase, and catalase were analyzed. Furthermore, histopathological evaluation of the tissues was performed.

Results:

The SCI in rats caused severe injury characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, lipid peroxidation, and increased oxidative stress. After SCI, tissue and serum IL-1β levels were significantly increased, but were significantly decreased by Anakinra administration. Following trauma, glutathione peroxidase, superoxide dismutase, and catalase levels were decreased; however, Anakinra increased the activity of these antioxidant enzymes. Malondialdehyde levels were increased after trauma, but were unaffected by Anakinra. Histopathological analysis showed that Anakinra effectively protected the spinal cord tissue from injury.

Conclusion:

Treatment with Anakinra reduces inflammation and other tissue injury events associated with SCI.Post-traumatic inflammatory reactions may play an important role in the secondary injury processes that occur after spinal cord injury (SCI).1,2 New treatment strategies like Anakinra aim to block or attenuate the critical mediators of inflammation in ischemia and reperfusion damage after spinal cord injuries. Primary traumatic mechanical injury to the spinal cord may cause neuronal death with irreversible recovery or regeneration. Neurons continue to die for several hours after SCI; however, this neuronal death could potentially be prevented. A large number of biochemical, and molecular cellular interactions result in secondary neuronal death. One of these interactions is the local inflammatory response in the injured spinal cord. It is thought that microglial cells might be the source of cytotoxic cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), which kill oligodendrocytes. Within one hour after SCI, increased synthesis, and/or secretion of IL-1β, is detectable at the injury site. Interleukin-1β is a member of the IL-1 cytokine family. The gene encoding this cytokine, and 8 other IL-1 family genes, form a cytokine gene cluster on chromosome 2.3 The mentioned cytokine is produced due to the activation of macrophages as a proprotein; the active form is produced secondary to the proteolytic action of caspase 1. The IL-1β is an important mediator of the inflammatory response, and is involved in a variety of cellular activities including cell proliferation, differentiation, and apoptosis.3 Anakinra is shown as an IL-1 receptor antagonist blocking the inflammation and cartilage degradation effects of naturally occurring IL-1 in rheumatoid arthritis, by competitively inhibiting the binding of IL-1 to the IL-1 type receptor.4 The IL-1 is produced in response to inflammatory stimuli and mediates various physiological responses including inflammatory and immunological reactions. In patients with rheumatoid arthritis, the natural IL-1 receptor antagonist is not found in sufficient concentrations in the synovium and synovial fluid to counteract the elevated IL-1 concentrations. Anakinra is considered a “biological response modifier” rather than a “disease-modifying antirheumatic drug” because it is able to selectively target the pathological elements of the disease.5 For this study, we determined the following endpoints of the inflammatory response: 1) histological damage, 2) cytokine expression (IL-1β), and 3) measurement of lipid peroxidation and oxidative stress (glutathione peroxidase [GPx], malondialdehyde [MDA], and superoxide dismutase [SOD]).6 The aim of the present study was to evaluate whether Anakinra administration could protect the spinal cord from lipid peroxidation and oxidative stress after SCI in rats.     

Overexpression of glucose-regulated protein 94 after spinal cord injury in rats

Glucose-regulated protein (GRP) 94 is a member of the stress protein family, which is localized in the endoplasmic reticulum (ER). Spinal cord injury (SCI) induced ER stress that results in apoptosis. However, the role of GRP94 in injury of the central nervous system remains unknown. In this study, we performed SCI in adult rats and investigated acutely the protein expression and cellular localization of GRP94 in the spinal cord. Western blot analysis revealed that GRP94 was low in normal spinal cord. It rose at 6h after SCI, peaked at 1 day, remained for another 3 days, then declined to basal levels at 5 days after injury. Immunohistochemistry further confirmed that GRP94 immunoactivity was expressed at low levels in gray matter and white matter in normal condition and increased after SCI. Double immunofluorescence staining showed that GRP94 was co-expressed with NeuN (neuronal marker), and GFAP (astroglial marker). In addition, caspase-12, caspase-3 and phospho-c-Jun NH2-kinase (p-JNK) levels increased at 6h, peaked at 1day, and then gradually reduced to normal levels for 2 weeks after SCI by western blot analysis. Co-localization of GRP94/caspase-12 and GRP94/p-JNK was detected in neurons and glial cells. Taken together, these data suggest GRP94 involvement in the injury response of the adult spinal cord of the rats.