The Expression of CUGBP1 After Spinal Cord Injury in Rats

Neurochemical Research - CUG-binding protein 1, a member of the CELF (CUGBP and embryonic lethal abnormal vision-like factor) family of RNA-binding proteins, is shown to be multifunctional,...     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

Regulation of Zinc Transporter 1 Expression in Dorsal Horn of Spinal Cord After Acute Spinal Cord Injury of Rats by Dietary Zinc

Zinc concentrations in the dorsal horn of spinal cord are important for wound healing, neurological function, and reproduction. However, the response of the spinal cord to alterations in dietary zinc is unknown in rats after spinal cord injury (SCI). The current study explored cellular zinc levels and zinc transporter 1 (ZnT1) expression in the dorsal horn of spinal cord with different dietary zinc after SCI. A hundred and forty-four male Wistar rats were randomly divided into four groups: sham-operated group (30?mg Zn/kg), zinc-high dietary SCI model group (ZH, 180?mg Zn/kg), zinc-adequate dietary SCI model group (30?mg Zn/kg), and marginal zinc-deficient dietary SCI model group (MZD, 5?mg Zn/kg). To test the hypothesis that dietary zinc may regulate role of ZnT1 expression in dorsal horn after acute SCI, we traced ZnT1 proteins and zinc ions with immunohistochemistry, western blot, and autometallography. Zinc and ZnT1 levels of the dorsal horn in ZH significantly increased after surgery (P?<?0.05), reached peak level (P?<?0.05) on the seventh day, and subsequently levels of their expression began to decrease. But zinc levels and ZnT1 expression of spinal cord in MZD dietary groups decreased (P?<?0.05) in SCI. There was a positive correlation between ZnT1 protein and zinc content in spinal cord (R?=?0.49880, P?=?0.0492). We found that both zinc and ZnT1 expressions in spinal cord are regulated by dietary zinc. These results indicate that dietary zinc may regulate the expression of ZnT1 in the dorsal horn of spinal cord after SCI. ZnT1 may, at the same time, play a significant role in the maintenance of zinc homeostasis in SCI.     

Spatiotemporal Pattern of RNA-Binding Motif Protein 3 Expression After Spinal Cord Injury in Rats

RNA-binding motif protein 3 (RBM3) belongs to a very small group of cold inducible proteins with anti-apoptotic and proliferative functions. To elucidate the expression and possible function of RBM3 in central nervous system (CNS) lesion and repair, we performed a spinal cord injury (SCI) model in adult rats. Western blot analysis revealed that RBM3 level significantly increased at 1 day after damage, and then declined during the following days. Immunohistochemistry further confirmed that RBM3 immunoactivity was expressed at low levels in gray and white matters in normal condition and increased at 1 day after SCI. Besides, double immunofluorescence staining showed RBM3 was primarily expressed in the neurons and a few of astrocytes in the normal group. While after injury, the expression of RBM3 increased both in neurons and astrocytes at 1 day. We also examined the expression profiles of proliferating cell nuclear antigen (PCNA) and active caspase-3 in injured spinal cords by western blot. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many RBM3-expressing cells and rare caspase-3 was observed in RBM3-expressing cells at 1 day after injury. Our data suggested that RBM3 might play important roles in CNS pathophysiology after SCI.     

LIN28 Expression in Rat Spinal Cord After Injury

LIN28, an RNA-binding protein, is known to be involved in the regulation of many cellular processes, such as embryonic stem cell proliferation, cell fate succession, developmental timing, and oncogenesis. However, its expression and function in central nervous system still unclear. In this study, we performed an acute spinal cord contusion injury (SCI) model in adult rats and investigated the dynamic changes of LIN28 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that LIN28 was present in normal spinal cord. It gradually increased, reached a peak at 3 day, and then nearly declined to the basal level at 14 days after SCI. Double immunofluorescence staining showed that LIN28 immunoreactivity was found in neurons, astrocytes and a handful of microglia. Interestingly, LIN28 expression was increased predominantly in astrocytes but not in neurons. Moreover, the colocalization of LIN28 and proliferating cell nuclear antigen was detected after injury. Western blot showed that LIN28 participated in lipopolysaccharide (LPS) induced astrocytes inflammatory responses by NF-κB signaling pathway. These results suggested that LIN28 may be involved in the pathologic process of SCI, and further research is needed to have a good understanding of its function and mechanism.     

Expression Profile and Role of EphrinA1 Ligand After Spinal Cord Injury

Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system’s response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.     

促凋亡基因Bax在大鼠脊髓损伤后的表达及意义

目的:研究促凋亡基因Bax表达与脊髓损伤(spinal cord injury,SCI)程度的关系.方法:Wistar大鼠36只,随机分成3组,为正常对照组、轻(中)度损伤组和重度损伤组.大鼠在脊髓损伤后14天处死,HE和Nissel染色观察脊髓组织形态结构和病理学变化,免疫组织化学S-P法检测脊髓中Bax表达情况.结果:Bax蛋白在大鼠脊髓损伤前后表达阳性率分别为5.6%和58.3%,有显著性差异(P＜0.05);轻(中)度脊髓损伤和重度脊髓损伤中的Bax的阳性率分别为18.5%和59.3%,有显著性差异(P＜0.05).结论:Bax基因表达与大鼠脊髓损伤有密切关系,且随着损伤程度加重Bax表达也增强.     

Decreased Expression and Role of GRK6 in Spinal Cord of Rats After Chronic Constriction Injury

Nerve injury and inflammation can both induce neuropathic pain via the production of pro-inflammatory cytokines. In the process, G protein-coupled receptors (GPCRs) were involved in pain signal transduction. GPCR kinase (GRK) 6 is a member of the GRK family that regulates agonist-induced desensitization and signaling of GPCRs. However, its expression and function in neuropathic pain have not been reported. In this study, we performed a chronic constriction injury (CCI) model in adult male rats and investigated the dynamic change of GRK6 expression in spinal cord. GRK6 was predominantly expressed in the superficial layers of the lumbar spinal cord dorsal horn neurons and its expression was decreased bilaterally following induction of CCI. The changes of GRK6 were mainly in IB4 and P substrate positive areas in spinal cord dorsal horn. And over-expression of GRK6 in spinal cord by lentivirus intrathecal injection attenuated the pain response induced by CCI. In addition, the level of TNF-α underwent the negative pattern of GRK6 in spinal cord. And neutralized TNF-α by antibody intrathecal injection up-regulated GRK6 expression and attenuated the mechanical allodynia and heat hyperalgesia in CCI model. All the data indicated that down-regulation of neuronal GRK6 expression induced by cytokine may be a potential mechanism that contributes to increasing neuronal signaling in neuropathic pain.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Spatiotemporal Patterns of SSeCKS Expression After Rat Spinal Cord Injury

Src suppressed C kinase substrate (SSeCKS) was identified as a PKC substrate/PKC-binding protein, which plays a role in mitogenic regulatory activity and has a function in the control of cell signaling and cytoskeletal arrangement. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the mRNA and protein expression and cellular localization of SSeCKS during spinal cord injury (SCI). Real-time PCR and Western blot analysis revealed that SSeCKS was present in normal whole spinal cord. It gradually increased, reached a peak at 3 days for its mRNA level and 5 days for its protein level after SCI, and then declined during the following days. In ventral horn, the expression of SSeCKS underwent a temporal pattern that was similar with the whole spinal cord in both mRNA and protein level. However, in dorsal horn, the mRNA and protein for SSeCKS expression were significantly increased at 1 day for its mRNA level and 3 days for its protein level, and then gradually declined to the baseline level, ultimately up-regulated again from 7 to 14 days. The protein expression of SSeCKS was further analysed by immunohistochemistry. The positively stained areas for SSeCKS changed with the similar pattern to that of protein expression detected by immunoblotting analysis. Double immunofluorescence staining showed that SSeCKS immunoreactivity (IR) was found in neurons, astrocytes, oligodendrocytes of spinal cord tissues within 5 mm from the lesion site. Importantly, injury-induced expression of SSeCKS was co-labeled by active caspase-3 (apoptotic marker), Tau-1 (the marker for pathological oligodendrocyte) and β-1,4-galactosyltransferase 1 (GalT). All the results suggested that SSeCKS might play important roles in spinal cord pathophysiology and further research is needed to have a good understanding of its function and mechanism. Feng Xiao and Min Fei contributed equally to this work.     

The Expression of VHL (Von Hippel-Lindau) After Traumatic Spinal Cord Injury and Its Role in Neuronal Apoptosis

The VHL (Von Hippel-Lindau) gene is a tumor suppressor gene, which is best known as an E3 ubiquitin ligase that negatively regulates the hypoxia inducible factor. The inactivation of VHL gene could result in the abnormal synthesis of VHL protein, which is in contact with the development and occurrence of renal clear cell carcinoma. However, the expression and possible function of VHL in central nervous system (CNS) is still unclear. To examine the function of VHL in CNS injury and repair, we used an acute spinal cord injury (SCI) model in adult rats. Western blot analysis showed an important upregulation of VHL protein, reaching a peak at day 3 and then declined during the following days. Double immunofluorescence staining showed that VHL was co-expressed with neurons, but not with astrocytes and microglia. Moreover, we detected that active caspase-3 had co-localized with VHL in neurons after SCI. Additionally in vitro, VHL depletion, by short interfering RNA, significantly reduced neuronal apoptosis. In conclusion, these data suggested that the change of VHL protein expression was related to neuronal apoptosis after SCI.

     

大鼠放射性脊髓损伤脊髓血流量变化规律

目的:放射性脊髓损伤(Radiation spinal cord injury,RSCI)是头颈部、胸部及上腹部肿瘤放射治疗和射线意外照射时的常见并发症,一般认为,白质坏死、脱髓鞘为其主要的病理学变化.然而,越来越多的证据表明血-脊髓屏障破裂和血管通透性增加等血管损伤远早于白质坏死和脱髓鞘改变.所以本文阐明大鼠放射性脊髓损伤病理生理过程中脊髓血流量变化规律.方法:将60只Sprague-Dawley (SD)大鼠随机分为12组,1组为对照,其余11组采用60Co放射治疗机行30 Gy大鼠颈髓C2-T2单次照射,剂量率为153 cGy/min,源皮距为80 cm,照射时长为1153 s,照射范围为2.0× 1.0 cm,对照组大鼠于麻醉后置于60Co放射治疗机下,佯照,照射前及照射后分别采用激光多普勒法测量脊髓血流量,11组大鼠于照射前以及照射后1、3、7、14、21、30、60、90、120、150、180天进行测量,以照射前测量值为基数,各时间点以基数的百分比表示该时间点脊髓血流量.结果:大鼠放射性脊髓损伤后,脊髓血流量在照射早期即有降低,照射后90天达到最低,随后脊髓血流量进入平台期.结论:阐明了大鼠放射性脊髓损伤后脊髓血流量的变化规律.大鼠放射性脊髓损伤可影响脊髓血流量,导致脊髓长期处于持续低灌流、缺血缺氧状态,最终导致脊髓不可逆性损伤.临床上放射性脊髓损伤的病人感到疲乏无力,出现神经系统的症状体征,通常死于脑疝.本文为临床上疲乏无力,出现神经系统的症状体征,死于脑疝放射性脊髓损伤的病人的早期防治提供病理生理基础.     

Serum Lipid Profile in Subjects with Traumatic Spinal Cord Injury

ResultsCases without preserved motor function (AIS A or B) had lower total and HDL cholesterol than the others (-11.4 [-21.5, -1.4] mg/dL total cholesterol and -5.1 [-8.8, -1.4] mg/dL HDL-c), and cases with all-limb involvement had lower total, HDL, and LDL cholesterol than those with only lower-limb involvement (-14.0 [-24.6, -3.4] mg/dL total cholesterol, -4.1 [-8.0, -0.2] mg/dL HDL-c, and -10.0 [-19.7, -0.3] mg/dL LDL-c) (all p<0.05). No association was found between lipid concentrations and time since injury. Concentrations of lipid subfractions and triglycerides in SCI subjects were lower than in sex- and age-stratified values from the reference sample.ConclusionA high degree of neurological involvement in SCI (anatomically higher lesions and AIS A or B) is associated with lower total cholesterol and HDL-c.     

HMGB1 Contributes to Regeneration After Spinal Cord Injury in Adult Zebrafish

High mobility group box 1 (HMGB1, also called amphoterin) facilitates neurite outgrowth in early development, yet can exacerbate pathology and inhibit regeneration by inducing adverse neuroinflammation when released from dying cells, suggesting that HMGB1 plays a critical, yet undefined role in neuroregeneration. We explored whether HMGB1 contributes to recovery after complete spinal cord transection in adult zebrafish. Quantitative PCR and in situ hybridization revealed that HMGB1 mRNA levels decreased between 12 h to 11 days after spinal cord injury (SCI), then returned to basal levels by 21 days. Western blot and immunohistological analyses indicated that the time course of HMGB1 protein expression after SCI parallels that of mRNA. Immunofluorescence staining revealed that HMGB1 translocates from nuclei into the cytoplasm of spinal motoneurons at 4 and 12 h (acute stage) following SCI, then accumulates in the nuclei of motoneurons during the ensuing chronic stage (after 6 days following SCI). Immunohistology of transgenic zebrafish, expressing green fluorescent protein in blood vessels, showed enhanced HMGB1 expression in blood vessels in the vicinity of motoneurons. Application of anti-sense HMGB1 morpholinos inhibited locomotor recovery by 34 % and decreased axonal regeneration by 34 % compared to fish treated with a control morpholino. The present study shows that HMGB1 expression increases in both endothelial cells and motoneurons, suggesting that HMGB1 promotes recovery from SCI not only through enhancing neuroregeneration, but also by increasing angiogenesis. The inflammatory effects of HMGB1 are minimized through the decrease in HMGB1 expression during the acute stage.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.