Protein Levels In Spinal Cord Research Articles

Pharmacodynamics assessment of β-carboline from the roots of Psammosilene tunicoides as analgesic compound

Ethnopharmacological relevanceThe root of Psammosilene tunicoides (W. C. Wu et C. Y. Wu) is a well-known medicinal herb for the treatment of pain, hemostasia and rheumatoid arthritis among Chinese people. Aim of the studyThe present study aimed to investigate the antinociceptive activity and mechanism of β-carboline alkaloids 1–4 which were extracted from the roots of P. tunicoides. Materials and methodsThe analgesic effects were evaluated using peripheral and central pain mouse models of nociception, including the formalin test and the tail flick test. The levels of glutamic acid (Glu) and nitric oxide (NO) in cerebellar cortexes and spinal cords (L4-6) were determined. The anti-inflammatory of all compounds were then assessed on RAW264.7 cells. ResultsOur results showed that compounds 1–4 had significant analgesic effects on both phases of formalin test of mice. Furthermore, all compounds showed suppressive effects on Glu in the brain and NO levels in the brain cortex and the spinal cord. Except for compound 1, the others could extend the pain threshold of hot water tail-flick in mice. In addition, compounds 2 and 3 (60 μmol/kg) could decrease GABAAα1 protein levels in spinal cord. All compounds exhibited anti-inflammatory effects by inhibiting lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells with half-maximal inhibitory concentration (IC50) 1.1–34.9 μM. Conclusionβ-carboline alkaloids from the roots of P. tunicoides had significant analgesic activity by both central and peripheral mechanisms. Our findings suggested that regulating the release of NO or Glu or GABAα1 are some of the mechanisms of analgesic activity of β-carboline alkaloids.

Abstract 872: Inhibition of Spinal Astrocytes Activation Attenuates Myocardial Ischemia-Reperfusion Injury by Regulating NGF/TRPV1 Nociceptive Signaling

Background and Aim: Spinal astrocytes activation are implicated in nociception and pain processing after noxious stimuli, while its role in cardiac ischemic nociception is unknown. We recently reported spinal nerve growth factor (NGF)-sensitized transient receptor potential vanilloid 1 (TRPV1) nociceptive signaling following cardiac ischemia and reperfusion (I/R). This study was design to investigate whether spinal astrocytes are activated following myocardial I/R and its effects on spinal NGF-TRPV1 signaling. Methods and Results: Myocardial I/R injury was induced by 30 min occlusion of the left coronary artery followed by 120 min of reperfusion in rats. Glial fibrillary acidic protein (GFAP), an astrocyte marker, was found up-regulated in thoracic spinal cord during ischemia and reperfusion. Intrathecally pre-injection of fluorocitrate (FC), an astrocyte inhibitor, reduced infarct size (IS/AAR, area at risk), from 48.4±2.67 to 29.7±3.13 (P<0.05, Fig.1 A). The high level of cardiac troponin T caused by I/R was decreased by FC injection as well (from 530±65 to 129±50 μg/μl, P<0.01, Fig.1 B). Upon immunofluorescence staining, GFAP immunopositive cells in spinal cord were found markedly increased after I/R, while the staining was suppressed by intrathecal FC administration (Fig.1 C). Moreover, the up-regualtion of NGF and TRPV1 protein levels in spinal cord were both inhibited by FC pretreatment (Fig.1 D-F). Conclusion: Myocardial I/R induced the activation of spinal astrocytes, inhibition of which reduces infarct size and cTnT level, as well as NGF-TRPV1 signaling, which suggests a novel therapeutic target for preventing myocardial ischemic injury.

Calpeptin is neuroprotective against acrylamide-induced neuropathy in rats

The aim of this study is to explore the potent neuroprotective effect of calpeptin (CP) on neuron damage induced by acrylamide (ACR) and its mechanism. Behavioural indicators such as hind limb splay, rota-rod performance, and gait analysis were assessed weekly to evaluate neurobehavioural changes after ACR and/or CP administration. The histopathological alterations and the changes of μ-calpain, m-calpain, microtubule-associated protein 2 (MAP2), and α-tubulin and β-tubulin protein levels in spinal cord were determined. Results showed that after administration of 30 mg/kg ACR, decreased body weight, attenuated neurobehavioural function, injury of motor neuron, increased protein levels of m-calpain and β-tubulin, suppressed MAP2 protein level, and no significant changes of μ-calpain and α-tubulin protein levels were observed compared with the control group rats. After administration of 200 μg/kg CP, partially restored body weight and neurobehavioural function, improvement of motor neuron injury, decreased protein levels of m- calpain and β-tubulin, and reversed effects of MAP2 protein level were observed compared with the ACR group rats. Our results suggested that CP alleviates neuropathy induced by ACR in rats. The calpain’s overactivation causes the degrading of MAP2 and eventually leads to the destruction of microtubules (MTs), which may be one of the mechanisms of cytoskeletal damage induced by ACR.

In vitro and in vivo effects of 2,4 diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Context-specific modulation of SMN transcript levels.

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.

Neuroprotective Effect of Calpeptin on Acrylamide-Induced Neuropathy in Rats.

Acrylamide (ACR) is a vinyl monomer with established human neurotoxic effects, which is characterized by the accumulation of neurofilaments (NFs) in the distal swellings of large axons in peripheral and central nervous systems. However, the mechanisms of neurotoxicity remain unclear. The objective is to investigate the neuroprotective effect of calpeptin (CP) on ACR-induced neuropathy and its mechanism. Female adult Wistar rats were randomly divided into four groups (control, CP, ACR, and ACR + CP group). Control group received 0.9 % saline, ACR and ACR + CP groups received 30 mg/kg ACR by intraperitoneal injection. In addition, CP and ACR + CP groups also received 200 µg/kg CP. Gait analysis and hind limb splay were measured weekly to analyze neurobehavioral changes. The calpain activity and the changes of NFs protein levels in spinal cord are determined. Compared with control group, body weight of rats in ACR group decreased by 11.3 % (P < 0.01), while in ACR + CP group body weight increased significantly by 8.3 % (P < 0.01) compared with ACR group by the end of the 4th week; gait score of rats in both ACR and ACR + CP groups increased significantly by 167 % and 100 % (P < 0.01) compared with control group, while it decreased significantly by 25.1 % (P < 0.01) in ACR + CP group compared with ACR group; the distance of hind limb splay in both ACR and ACR + CP groups increased by 76.7 % and 49.5 % (P < 0.01) compared with control group, while it decreased by 15.4 % (P < 0.01) in ACR + CP group compared with ACR group; calpain activity of spinal cord at ACR and ACR + CP groups increased significantly by 14.9 % and 10.0 % (P < 0.01) compared with control group, while it decreased 4.2 % (P < 0.01) in ACR + CP group compared with ACR group; compared with control group, the levels of light NF (NF-L), medium NF (NF-M) and heavy NF (NF-H) subunits increased by 81.2 %, 263.6 % and 22.6 % (P < 0.01) in the supernatant of ACR group in spinal cord tissue and increased by 28.4 %, 96.6 % and 10.6 % (P < 0.01) in ACR + CP group, while the levels of NF-L, NF-M and NF-H subunits decreased by 29.1 %, 45.9 % and 9.8 % (P < 0.01) in ACR + CP group compared with ACR group. The present results suggested that CP can relieve ACR neuropathy by decrease calpain activity and NFs degradation. The changes of calpain activity and NFs may be one of the mechanisms of ACR-induced neuropathy.

Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T-cell death-associated gene 8 (TDAG8) has been shown to participate in complete Freund's adjuvant-induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element-binding (pCREB) protein levels were up-regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain-induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H-89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

Increase in Trx2/Prx3 redox system immunoreactivity in the spinal cord and hippocampus of aged dogs

We previously reported that no distinct neuronal loss occurred in the aged dog spinal cord, although oxidative stress was increased in the aged dog spinal cord. Thioredoxin 2 (Trx2)/peroxiredoxin 3 (Prx3) redox system is a major route for removing H 2O 2 in the central nervous system. In the present study, we compared the distribution and immunoreactivity of thioredoxin reductase 2 (TrxR2), Trx2 and Prx3 and their protein levels in the spinal cord and hippocampus between the adult (2–3 years) and aged (10–12 years) dogs. The number of TrxR2-immunoreactive neurons was slightly increased; however, its immunoreactivity was significantly increased in the aged spinal cord compared to that in the adult spinal cord. On the other hand, the number and immunoreactivity of both Trx2- and Prx3-immunoreactive neurons were significantly increased in the spinal cord of the aged dog. Similarly, in the hippocampus of the aged dog, TrxR2, Trx2 and Prx3 immunoreactivity and protein levels were markedly increased compared to those in the adult dog. These results indicate that the increases of TrxR2, Trx2 and Prx3 immunoreactivity and their protein levels in the aged spinal cord and hippocampus may contribute to reducing neuronal damage against oxidative stresses during normal aging.

Activation of Matrix Metalloproteinases-9 after Photothrombotic Spinal Cord Injury Model in Rats

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9 have been known to play an important role in secondary inflammatory reaction after spinal cord injury (SCI). The aim of this study was to investigate the expression and activity of MMP-2 and MMP-9 and to determine their relationship with disruption of endothelial blood-barrier after photochemically induced SCI in rats. Female Sprague-Dawley rats, weighing between 250 and 300 g (aged 8 weeks) received focal spinal cord ischemia by photothrombosis using Rose Bengal. Expressions and activities of MMP-2 and MMP-9 were assessed by Western blot and gelatin zymography at various times from 6 h to 7 days. Endothelial blood-barrier integrity was assessed indirectly using spinal cord water content. Zymography and Western blot analysis demonstrated rapid up-regulation of MMP-9 protein levels in spinal cord after ischemic onset. Expressions and activities of MMP-9 showed a significant increased at 6 h after the photothrombotic ischemic event, and reached a maximum level at 24 h after the insult. By contrast, activated MMP-2 was not detected at any time point in either the experimental or the control groups. When compared with the control group, a significant increase in spinal cord water content was detected in rats at 24 h after photothrombotic SCI. Early up-regulation of MMP-9 might be correlated with increased water content in the spinal cord at 24 h after SCI in rats. Results of this study suggest that MMP-9 is the key factor involved in disruption of the endothelial blood-barrier of the spinal cord and subsequent secondary damage after photothrombotic SCI in rats.

Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality. SMA is caused by loss of functional survival motor neuron 1 (SMN1), resulting in death of spinal motor neurons. Current therapeutic research focuses on modulating the expression of a partially functioning copy gene, SMN2, which is retained in SMA patients. However, a treatment strategy that improves the SMA phenotype by slowing or reversing the skeletal muscle atrophy may also be beneficial. Myostatin, a member of the TGF-beta super-family, is a potent negative regulator of skeletal muscle mass. Follistatin is a natural antagonist of myostatin, and over-expression of follistatin in mouse muscle leads to profound increases in skeletal muscle mass. To determine whether enhanced muscle mass impacts SMA, we administered recombinant follistatin to an SMA mouse model. Treated animals exhibited increased mass in several muscle groups, elevation in the number and cross-sectional area of ventral horn cells, gross motor function improvement and mean lifespan extension by 30%, by preventing some of the early deaths, when compared with control animals. SMN protein levels in spinal cord and muscle were unchanged in follistatin-treated SMA mice, suggesting that follistatin exerts its effect in an SMN-independent manner. Reversing muscle atrophy associated with SMA may represent an unexploited therapeutic target for the treatment of SMA.

Spinal CK2 regulates nociceptive signaling in models of inflammatory pain

Casein kinase 2 (CK2) is a widely expressed protein kinase. Over the last several years a long list of protein substrates has evolved, many of which have proven or hypothesized roles in nociceptive signal transmission. However, CK2 has not itself been demonstrated to participate in nociception prior to this time. We set out to test the hypothesis that spinal CK2 regulates nociception using several pain models. Our first studies focused on the ability of the selective CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBBT) and 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) to reduce formalin-stimulated pain behaviors in mice. Both phases of the response to subcutaneous formalin were strongly inhibited by intrathecal administration of TBBT or DRB in dose-dependent fashion. Likewise, using the complete Freund's adjuvant (CFA) model of chronic inflammatory pain, TBBT was observed to strongly reduce mechanical allodynia. The inhibition of spinal CK2 with either inhibitor did not, however, alter withdrawal latencies in the hotplate thermal pain model while intrathecal morphine was very effective. Immunohistochemical studies demonstrated all three known CK2 subunits, α, α′ and β to be expressed in spinal cord tissue as did real-time PCR experiments. While mRNA levels for each of the subunits was transiently enhanced after formalin or CFA hindpaw injection, overall spinal cord protein levels were not elevated in a sustained fashion. Our results indicate that CK2 participates in inflammatory nociception both in the acute and chronic phases. Simple changes in the abundance of spinal CK2 subunits do not likely underlie these phenomena, however.

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1beta-induced neutrophil recruitment.

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT-PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1beta (IL-1beta) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2alpha, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1beta induced greater CINC-1 and CINC-2alpha expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2alpha protein were observed in response to IL-1beta injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2beta mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.

Ciliary neurotrophic factor receptor expression in spinal cord and motor cortex in amyotrophic lateral sclerosis

Ciliary neurotrophic factor (CNTF) is known to rescue motor neurones in animal models of injury and neurodegeneration and we have recently described regional changes of CNTF protein levels in spinal cord from patients with sporadic motor neurone disease of the amyotrophic lateral sclerosis (ALS) type. However, information is lacking about the CNTF receptor in this condition. We have therefore studied mRNA levels for the α subunit of the CNTF receptor (CNTFRα) and for CNTF itself in postmortem spinal cord and cerebral cortex in patients with sporadic ALS and matched controls. We report that in the spinal cord of ALS patients there is a marked increase in the hybridisation signal for CNTFRα subunit mRNA overlying motor neurones. In contrast, very little mRNA for CNTFR α subunit was found in the motor cortex and no differences were seen between ALS and controls. We were unable to detect any hybridisation signal for CNTF mRNA. Our findings provide evidence for regional differences in CNTF receptor expression in upper- and lower-motor neurones in ALS.