The Effect Of NG2 Cells In Spinal Cord Of Neuropathic Pain In Rats

BackgroundNeuropathic pain refers to chronic pain that originates from pathology of the nervous system. The International Association for the Study of Pain (IASP,1994) defines neuropathic pain as "pain initiated or caused by a primary lesion or dysfunction of the nervous system". Infection (herpes zoster), nerve trauma, metabolic diseases, cancer chemotherapy, surgery, radiation, neurotoxicity drugs, nerve compression, inflammation and tumor invasion are examples of diseases that may cause neuropathic pain. Acute pain plays a warning and "protective" role. By contrast, neuropathic pain can persist and serves no known defensive, or other helpful, function. And it might even affect the quality of life seriously. The mechanism of neuropathic pain is very complex. There are a series of complex changes in the nervous system from nerve damage (nociceptive stimulus) to pain production. The spinal cord is the primary center of the pain information delivery and integration. The spinal dorsal horn includes different peripheral afferent nerves, descending projection fibers from the brain stem and cerebral cortex and local interneurons. These compose a very complex neural network, and it is very rich in bio-active substances. Therefore, the spinal cord plays an important role in the development of neuropathic pain. Traditional view is that neurons are important signal cells in the central nervous system, and the role of glial cells is only insulation, nutrition and support. Therefore, a large number of experiments in pain research focused on the function of neurons in the past. But in recent years, we find that glial cells play an important role in the process of pain. Colburn proposed that microglia are more important for the initiation, while astrocytes are more important for the maintenance of neuropathic pain. Injury to a peripheral nerve initiates increased release of neurotransmitters such as substance P, glutamate and possibly ATP from the central terminals of primary afferents. These neurotransmitters can activate glial cells in the spinal cord through binding to their receptors. These triggers cause production and release of a large number of pain-related mediators from glial cells, such as cytokines, inflammatory mediators and neurotrophins. These agents are then capable of further enhancing neurons and glial cells activation, eventually leading to the generation and expansion of pain signals, thereby contributing to neuropathic pain.Recent studies suggest that there is a class of cells with both neuronal and glial properties in central nervous system. These cells show specific expression of NG2 surface chondroition sulphate proteoglycan on their surface, and are known as NG2 cells. The latest results indicate that NG2 cells are a population of CNS glial cells that are distinct from mature oligodendrocytes, astrocytes and microglia. These cells widely distribute in the gray matter and white matter. The most common phenomena indicating NG2 cells response to stimulation are up-regulation of chondroitin sulfate proteoglycan expression, increase in cell size (hypertrophy) and increase in the number of cellular processes in a variety of nervous system injury and disease. The discovery of expressions of glutamate and y-aminobutyric acid (GABA) receptors in NG2 cells and the formation of direct synaptic junctions between NG2 cells and glutamatergic and GABAergic neurons in the hippocampus implied they are integrated in the neural network. NG2 cells can produce and release a variety of neural excitatory substances in the course of nervous system diseases. In addition, lipopolysaccharide (LPS) induced the expression of NG2 and cytokines in microglial cells. However in this study, LPS did not induce the mRNA expression of cytokines in microglial cells transfected with NG2 siRNA. These studies suggest that NG2 cells might be involved in neuropathology, including the development of pain. However, we are still far from a complete understanding of the functional roles they play in the CNS.Based on the above theory and research base, our objective is to study the effect of NG2 cells in spinal cord in a rat model of neuropathic pain through the following three-part: 1. After the rat models of spinal nerve ligation (SNL) for neuropathic pain were made, the changes of nociceptive behaviors, including mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD), were observed. And then, the changes of NG2 cells and the expression of NG2 molecule in spinal cord were detected.2. The rat NG2 cells were cultured in vitro. The expression of NG2 and pain-related molecules were detected after activation of the membrane surface AMPA receptor. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed and its interference effect was identified. The changes of the expression of pain-related molecules were detected after activation of the membrane surface AMPA receptor when NG2 molecule was down-regulated.3.The lentiviral vectors for RNA interference of the rat NG2 gene were injected intrathecally, the change of the expression of pain-related molecules in NG2 cells were detected and the nociceptive behaviors were observed. Methods and Results1:The change of NG2 cells in spinal cord of nuropathic pain in ratsMethods:Ninety-six female Sprague-Dawlye rats were selected and randomly divided into three groups:Naive group (not received any surgery, n=24); Sham group (the rat L5 spinal never was exposed, n=24) and SNL group (the rat L5 spinal never was exposed and ligated, n=48). Mechanical witndrawal threshold and thermal withdrawal duration were used to measure the response of operative hind paw to mechanical and radiant heat stimulation before operation and 3d,7d,14d,21d,28d after operation. At the same time, the rats were perfused with PBS followed by 2% paraformaldehyde (PFA) in PBS (4 rats every time point). The lumbar 4-6 spinal cord frozen slices were made and the changes of NG2 cells were observed through immunohistochemistry. Meanwhile the western blot analysis method was also used to detect the expression of NG2 molecule in spinal cord of the rats.Results:The mechanical witndrawal threshold and thermal withdrawal duration had not any difference among all groups before operation (P>0.05). The mechanical witndrawal threshold and thermal withdrawal duration in sham group and naive group had not significantly difference between before and after operation (P>0.05). The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group at 3d,7d,14d,21d,28d after operation compare to before operation (P<0.05). The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group at 3d,7d,14d,21d,28d after operation compare to sham group (P<0.05). The immunohistochemistry result displayed that the cell size of NG2 cells and the number of cellular processes were increased at 7d,14d and 21d after operation. The number of NG2 cells in the lumbar 4-6 spinal dorsal horn was no difference between before and after operation. Meanwhile the western blot result revealed that the expression of NG2 molecule in lumbar 4-6 spinal cord was significantly increased at 7d,14d and 21d after operation in SNL group (P<0.05).2:The change of expression of pain-related molecules in NG2 cells after activation of AMPA receptor in vitroMethods:NG2 cells were isolated from neonatal rat hippocampus by Percoll gradient methods and cultured. NG2 cells were identified by immunocytochemistry and the purity of cells was detected by fluorescence-activated cell sorting. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed and its interference effect was identified. NG2 cells were cultured in 6-well plates and divided into three groups:control group (treated without any agents, n=12), AMAP group (treated with AMPA receptor agonist AMPA 0.5mM, n=12), AMPA+CNQX group (treated with AMPA receptor agonists AMPA 0.5mM and AMPA receptor antagonist CNQX 50μM, n=12). The expression of NG2 molecule, neurotrophic factor BDNF, NGF and cytokines IL-1(3, TNF-a were detected by Real-time PCR method at 1h,6h,12h,24h after treated (3 wells per time point). NG2 cells were cultured in 6-well plates and divided into two groups:control group (treated with AMPA receptor agonist AMPA 0.5mM at 4d after with LV-Con-RNAi treatment, n=3), experimental group (treated with AMPA receptor agonist AMPA 0.5mM at 4d after with LV-NG2-RNAi treatment, n=3). The change of the expression of NG2 molecule, neurotrophic factor BDNF, NGF and cytokines IL-1(3, TNF-a were detected by Real-time PCR method at 12h after treated. Results:Rat NG2 cells were cultured sucssesfully in vitro.Lentiviral vector LV-NG2-RNAi was constructed and the titer was 8×108TU/ml.The interference efficiency was up to 80% when C6 cells were infected with MOI=20.Real-time PCR results showed that the expression of NG2,BDNF and NGF were increased at 1h,6h,12h,24h after activation of AMPA receptor, there was significant defference between control and AMPA group (P<0.05); the expression of IL-1βwere increased at 6h,12h,24h after activation of AMPA receptor, there was significant defference between control and AMPA group (P< 0.05);the expression of TNF-a was increased at 1h,6h,12h after activation of AMPA receptor, there was significant defference between control and AMPA group (P<0.05). Real-time PCR results showed that the expression of NG2 molecule was decreased in experimental group, there was significant defference between control and experimental group (P<0.05);the expression of BDNF, NGF, IL-1βand TNF-αwere decreased in experimental group, there was significant defference between control and experimental group (P<0.05).3:The effect of intrathecal injection of NG2-shRNA lentivirus of nuropathic pain in ratsMethods:Forty-eight female Sprague-Dawlye rats were selected and randomly divided into four groups:Naive group (not received any surgery, n=12);SNL group (noly received L5 spinal nerve ligation surgery, n=12);SNL+Con group (received SNL surgery at 7d after intrathecal injection of LV-Con-shRNA lentivirus, n=12);SNL+NG2 group (received SNL surgery at 7d after intrathecal injection of LV-NG2-shRNA lentivirus, n=12).The western blot analysis method was used to detect the expression of NG2 molecule in spinal cord of the rats. Meanwhile the expression of neurotrophic factor BDNF, NGF and cytokines IL-1β, TNF-a in NG2 cells were detected by fluorescence-activated cell sorting method. Mechanical witndrawal threshold and thermal withdrawal duration were used to measure the response of operative hind paw to mechanical and radiant heat stimulation before operation and 3d,7d, 14d,21d,28d after operation.Results:The expression of NG2 molecule in spinal cord of the rats in SNL group and SNL+Con group was increased (P<0.05), but the expression of NG2 molecule in SNL+NG2 group had not defference compare to Naive group (P>0.05). The expression of BDNF and NGF in NG2 cells was increased in SNL group and SNL+Con group, but there was no defference between SNL+NG2 group and Naive group. The expression of IL-1βand TNF-αin NG2 cells was increased in SNL group, SNL+Con group and SNL+NG2 group. The mechanical witndrawal threshold was decreased and thermal withdrawal duration was increased in SNL group and SNL+Con group 3d after operation (P<0.05).The mechanical witndrawal threshold in SNL+NG2 group was significantly decreased 14d after operation (P<0.05). The thermal withdrawal duration in SNL+NG2 group was significantly increased 7d after operation (P<0.05).4:Statistical analysisAll of the analyses were performed by SPSS 13.0 software package. Data are expressed as mean±standard error of the mean (SEM). The data were analyzed by repeated measures analysis of variance or one-way analysis of variance. A P value of<0.05 was accepted as significant. Major Results:1.The rat models of spinal nerve ligation(SNL) for neuropathic pain were made successfully through observing the change of nociceptive behaviors. The immunohistochemistry result displayed that NG2 cells in lumbar 4-6 spinal cord were activated in rat SNL model, the cell size of NG2 cells and the number of cellular processes were increased. Meanwhile the western blot result revealed that the expression of NG2 molecule in spinal cord was significantly increased.2. The rat NG2 cells were cultured successfully in vitro. The expression of NG2 and pain-related molecules were increased after activation of membrane surface AMPA receptor. Lentiviral vector of shRNA based on rat NG2 gene sequence was constructed successfully. The expression of pain-related molecules was decreased after activation of membrane surface AMPA receptor when NG2 molecule was down-regulated.3. The expression of NG2 molecule in spinal cord of the rats suffering with neuropathic pain was decreased after intrathecal injection of the lentiviral vector for RNA interference of the rat NG2 gene. The expression of BDNF and NGF in NG2 cells in lumbar 4-6 spinal cord was decreased. The onset of both thermal hyperalgesia and mechanical allodynia was delayed.Major Conclusion:1.NG2 cells in spinal cord are activated in rat SNL model and involved in the processes of pain. 2. NG2 and pain-related molecules are upregulated after activation of the membrane surface AMPA receptor. NG2 molecules are involved in the expression of pain-related molecules in NG2 cells after activation of the membrane surface AMPA receptor.3.The expression of BDNF and NGF in NG2 cells in spinal cord of the rats suffering with neuropathic pain was decreased after intrathecal injection of the lentiviral vector for RNA interference of the rat NG2 gene. And the onset of pain was delayed.Innovation:1.Recent studies have found that NG2 cells are involved in a variety of nervous system injury and disease. In this study, we observed the changes of NG2 cells and the expression of NG2 molecule in spinal cord of the rats suffering with L5 spinal nerve ligation firstly, and confirmed that NG2 cells are involved in the processes of pain.2.Evidence show that NG2 cells have AMPA-type glutamate receptors which are activated by neural activity and result in raised intracellular calcium. But now there are not experiments about the subsequent biological effects, including gene expression in cells. In this study, we observed the expression of NG2 and pain-related molecules after activation of membrane surface AMPA receptor and revealed the relationship between NG2 molecule and these pain-related molecules.Prospect:1.In this study, we found that NG2 cells in spinal cord were activated in rats suffering with neuropathic pain; the expression of NG2 and pain-related molecules were upregulated after activation of membrane surface AMPA receptor; the expression of pain-related molecules were decreased after activation of membrane surface AMPA receptor when NG2 molecules were down- regulated. But the signal pathway between NG2 molecule and pain-related molecules still need further study.2.Recent studies indicated NG2 cells formed direct synaptic junctions with neurons. Nociceptive signals activate NG2 cells through synaptic connections, and activated NG2 cells release pain-related molecules. These molecules might promote the spinal dorsal horn neurons sensitized furtherly. Therefore, the relationship between NG2 cells and neurons in spinal cord need to study.