ABSTRACT
Objective To explore the effects of melatonin (MT) on the expression of interleukin (IL)-10,interleukin-6 and interleukin-8 as well as the inflammatory reaction and nerve repair after acute spinal cord injury (SCI).Methods One hundred and eight Sprague-Dawley rats were randomly divided into a spinal cord injury group (group A),an MT treatment group (group B) and a sham operation group (group C),each with 36 rats.SCI models were established in the rats of groups A and B using a version of Allen's weight drop method (50gcf at the T12 level).Group C had removal of the lamina only.Ten minutes later,group A was injected with 5％ ethanol in saline (the MT solvent) and group B with 100 mg/kg of melatonin preparation.At 6,12,18 and 24 hours,IL-6,IL-8 and IL-10 levels in serum were detected in 6 rats of each group.At 18 hours post-surgery,spinal cord specimens were taken from 6 rats of each group for hematoxylin eosin staining,morphological examination and immunohistochemical SP detection of IL-10 expression.Results The specimens of group A showed inflammatory reaction and ulceration at 48 h; groups B and C had no ulcers.Group B showed the highest levels of IL-10 in serum and IL-10 mRNA in the spinal cord,while group C showed the lowest level.The differences were statistically significant.Group A had the highest levels of IL-6 and IL-8 and group C had the lowest.The difference between group B and groups A and C was significant.The morpho-logical observation showed that after melatonin treatment the IL-10 levels in the spinal cord's central canal and around the gray matter improved.Conclusions Melatonin can improve nerve lipid peroxidation and inflammatory reaction in the treatment of spinal cord injury by increasing IL-10 expression and inhibiting IL-6 and IL-8 expression.
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Three myelin proteins, Nogo-A, MAG and OMgp, transduce their neurite-outgrowth inhibitory signal through a common receptor complex: NgR/ p75NTR (or TROY). Recently, LINGO-1 is identified as another essential component and regulator for the Nogo-66 receptor/p75 signaling complex. LINGO-1 is restricted to express in CNS, neuronal LINGO-1 is shown to be involved in the signal transduction from three myelin proteins, and Lingo-1 in oligodendrocyte negatively regulates the differentiation and myelination of oligodendrocyte. To investigate the potential activity of LINGO-1 in neuronal apoptosis, LINGO-1-Fc fusion protein including the extracellular LRR and IgC2 domain, was used as functional antagonist to study its protective effect on low-potassium induced apoptosis of cerebellar granule neurons (CGNs). In judgement of the apoptotic nuclei stained by Hoechst, LINGO-1-Fc pretreatment for 2 h significantly prevents apoptosis of CGNs. Although GST-LINGO-1 protein, including the LRR domain, binds to the CGN cultures in the same way with LINGO-1-Fc, it doesn't prevent the apoptosis of CGNs. These results indicate that LINGO-1-Fc fusion protein prevents low-potassium induced apoptosis of cerebellar granule neurons in certain conditions and this activity is probably IgC2 domain dependent.
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Objective To detect Nogo-A proteolytic fragment in CNS injury models and investigate its proteolytic mechanism as well as potential functions. Methods Western blot was performed to detect Nogo-A proteolytic fragment after spinal cord transection,brain ischemia and KA-induced cerebral cortex injury in rats. Results We detected Nogo-A proteolytic fragment in spinal cord transection and brain ischemia models,whereas no Nogo-A proteolytic fragment was found in KA-induced cerebral cortex injury model.Conclusion\ CNS mechanical and ischemic injury may induce the degradation of Nogo-A by certain mechanisms.
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Objective To investigate the expression pattern and possible function of Nogo-A during neuronal growth. Methods E18 rat hippocampal neurons were primarily cultured both in high-density and low-density conditions. Immunohistochemistry and Western blot were performed to detect Nogo-A expression and distributional changes. Results Nogo-A was found in hippocampal neurons, mainly located in the cytoplasm, plasma membrane and neurites. It was detected at the proximal part of all neurites before axon formation. In axons, Nogo-A was enriched in the distal segment and axonal growth cone. In mature neurons, the fiber net work displayed a large number of Nogo-A immunoreactive varicosities. Conclusion The present results indicate that the neuronal Nogo-A may be involved in the process of neurite outgrowth and axonal projection.
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Objective To explore the effects of expressing human ciliary neurotrophic factor (hCNTF) mediated by retroviral vector in olfactory ensheathing cells(OECs) on the survival and neurite outgrowth of cultured neurons. Methods S\|hCNTF fragment was digested with endonucleases(Kpn I and Xba I) from pcDNA\-3\|S\|hCNTF plasmid and cloned into pRev\|TRE vector.The harvested pRev\|TRE\|hCNTF was identified and transfected with pRev\|Tet\|On into ecotropic Ecopack\|293 cells,resulting in 2 retroviral supernatants(pRev\|TRE\|hCNTF and pRev\|Tet\|On).Primarily cultured rat olfactory ensheathing cells(OECs) were co\|infected with the 2 retroviruses,and induced to secrete hCNTF with different concentrations of doxycline.The secreted hCNTF in OEC culture supernatant was detected with Western\|blot.Dorsal root ganglion (DRG) from a postnatal rat of 2 days was co\|cultured with CNTF\|modified OECs,and the supernatant was used to culture retinal ganglion cells(RGCs).Following ?\|tubulin immunocytochemical staining,the length of DRG neurites were measured,while the numbers of surviving RGCs were counted. Results 1.Individual 630bp and 400bp fragments were digested from pRev\|TRE\|S\|hCNTF expression vector with endonucleases(Hind Ⅲ and BamH Ⅰ),and respected direction and integration of hCNTF cDNA which inserted pRev\|TRE vector were identified; 2.The expression of 24kD CNTF proteins in CNTF\|modified OEC culture supernatant was positively\|correlated with the concentration of doxycline,while no such protein expression was detected in the control groups; 3.The number of surviving RGCs in CNTF\|modified OECs group(41^34?5^4) was significantly higher than those in unmodified OEC(23^15?4^7),OECs(24^55?5^8) and blank(16^8?6^5) groups;and 4^The neurites of DRG were longer (660?67?m) and denser in CNTF\|modified OECs group,as compared with unmodified OECs(418?45?m),Mock+OECs(400?65?m) and blank (0?m) control groups.No process migrated and grew from the tissue mass in blank group.Conclusion\ hCNTF can be expressed in OECs with a doxycline concentration\|dependent manner after transfected via pRev\|TRE\|S\|hCNTF vector,and possesses a marked enhancing effect on the survival and neurite outgrowth of cultured neurons.[