The Identification And Analysis Of Neural Nitric Oxide Synthase Diversities In Spinal Cord In Rats With Neuropathic Pain

Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. The reasons responsible for neuropathic pain include trauma, ischaemic injury, infection/inflammation, cancer, drugs and compression etc. Patients with neuropathic pain do not respond to non-steriodal anti-inflammatory drugs and have resistance or insensitivity to opiates. The current pharmacological mainstays of clinical management are tricyclic anti-depressants and certain anticonvulsants, but these only achieve clinical significant (greater than 50%) pain relief in less than 50% of patients and are associated with sub-optimal side effect profiles.The mechanisms leading to neuropathic pain are not clear. The majority of research associated with neuropathic pain mechanisms has concentrated on changes in the peripheral nerve or spinal cord after peripheral never injury, therefore, according to most available evidence relating to changes in these parts of the nervous system, the mechanism were divided into peripheral and central mechanism. There also is a concept developed to reveal the mechanism of neuropathic pain, which considered neuropathic pain as plasticity course composed of activation, modulation and modification terms.The mechanisms of neuropathic pain are so complicated in spinal cord. Drugs that target the NO/cGMP signaling pathway show controversial results in treatment of neuropathic pain. On one hand, NO precursor (L-arginine) and NO donors (sodium nitroprusside, S-nitroso-N-acetylpenicillamine) potentiated the hyperalgesia and allodynia significantly suggesting proalgesic effect in neuropathic rats; on other hand, the analgesia of intrathecal clonidine or neostigmine depend on NO/cGMP pathway and antinociceptive effect can be eliminated by NOS inhibitors.To elucidate the controversial results about NOS, This study tries to select a proper animal model for neuropathic pain to detect the changes in NOS expression and activity, and then concentrate on one kind of NOS to explain the complicated mechanism about NOS.Methods1 The most suitable animal model of neuropathic pain was selected in this study through comparing the sensibility to pain stimulation in three neuropathic pain models, chronic constriction injury of sciatic nerve (CCI), spinal nerve ligation model (SNL) and spared nerve injury (SNI). Then analyze the mechanism leading to neuropathic pain in rat spinal cord. 19 female SD rats (weight ranged from 150g to 200g) were randomized into four groups (control group, CCI group, SNL group, and SNI group). The sciatic nerve of the left hind leg was exposed without any injury in control group, subsequently the incision was sutured immediately. CCI group was prepared for CCI according to the method described by Gary J. SNL group for SNL according to Sun, and SNI group for SNI according to Isabelle. During the development of allodynia and hyperalgesia, Touch stimulator and radiant heat stimulator were used to measure the response of operative hind paw to mechanical and radiant stimulation. On the 15th day after operation, the animals were anesthetized with intraperitoneal injection of 10% chloral hydrate 400 mg·kg^-1. The L_5,6 segment of the spinal cord was harvested. Neuronal, astrocytic and microglial activation was determined by immuno-histochemistry with antibodies of C-Fos (a proto-oncogene protein), GFAP (an astrocyte marker) and OX-42 (a microglial marker).2 Western blotting and immunohistochemistry were performed to detect the activity of MEK-ERK and NF-kappa B signaling pathway in rat spinal cord. 12 female SD rats (weight ranged from 150 to 200g) were randomized into two groups, control group and Group SNI. No any nerve injury was applied for control group. Surgery was performed for SNI neuropathic pain model in group SNI. Foot-lift response frequency to mechanical stimulation for ipsilateral hindpaw was assessed by 12g and 2g touch stimulator. On the 11th day after operation, 3 rats from each group were fixed by perfusion for immunohistochemistry. Then proteins from ipsilateral L4-6 spinal cord in other 3 rats in each group were extracted for western blot analysis. Western blotting and immunohistochemistry were performed with antibodies specific for MEK, p-MEK, p-ERK and NF-kappa B.3 The pattern of expression and activity about three main nitric oxide synthases in spinal cord of rat with neuropathic pain were determined to evaluate their potential in modulating of pain. 20 female Sprague-Dawley rats were randomized into two groups (control group and Group SNI). Each consists of 10 rats. The rats of control group had a sham surgery on left hind limb. Group SNI had a partial lesion on left sciatic nerve (Spared Nerve Injury). After neuropathic pain developing, all rats were killed by cervical dislocation. L_4～6 spinal cord segments was derived into left and right side and retrieved respectively. The operative side of spinal cord segment in control group, and two sides of spinal cord segment in Group SNI were store at -80℃ for next experiments. Total RNA in 5 rats in each group was respectively extracted from rat spinal cord for RT-PCR of nNOS, eNOS and iNOS with 6-actin as an internal standard. The left spinal cord segments were homogenized to extract protein for determination of total NOS, cNOS and iNOS activity.4 The expression profiles of PSD95, NR2B and nNOS were detected to identify if there is coordination between NR2B and nNOS. 12 female SD rats (weight ranged from 150 to 200g) were randomized into two groups, control group and Group SNI. Control group was taken as a control group without any nerve injury. Surgery was performed for SNI neuropathic pain model in Group SNI. Foot-lift response frequency to mechanical stimulation for ipsilateral hindpaw was assessed by 12g and 2g touch stimulator. On the 11th day after operation, 3 rats from each group were fixed by perfusion for immunohistochemistry to analyse the expressions of NR2B and nNOS. Total RNA in ipsilateral L4-6 spine from Other 3 rats in each group were reverse-transcribed and amplified by PCR to determine the expression of PSD95 in mRNA level.5 The diversity and changes in nNOS was analyzed in spinal cord of rats with neuropathic pain. 20 female SD rats (weight ranged from 150 to 200g) were randomized into two groups, control group and Group SNI. Control group was taken no any nerve injury. Surgery was performed for SNI neuropathic pain model in Group SNI. Foot-lift response frequency to mechanical stimulation for ipsilateral hindpaw was assessed by 12g and 2g touch stimulator. On the 11th day after operation, 3 rats from each group were fixed by perfusion for immunohistochemistry to analyse the expression of nNOS. Proteins from ipsilateral L4-6 spinal cords from other 3 rats in each group were detected with an antibody specific for COOH-terminal domain of nNOS by methods of Western blot. Total RNA were extracted from ipsilateral L4-6 spinal cord in other 4 rats in each group. Rapid amplification of 5'-RACE-PCR was performed to identify the diversity of 5'terminus of nNOS, and then southern blot confirmed them. RT-PCR devised for different 5'terminus of nNOS was amplified to test presence of the diversity of nNOS in mRNA.6 The effects of overexpression of nNOS α or nNOS β on NF-kappa B activity and apoptosis were determined in PC12 cells. PC12 cell was transfected with pcDNA3-nNOS α or pcDNA3-nNOS β . The expression efficiency of nNOS was detected by immunohistochemistry and western blot analysis with an antibody specific for the COOH-terminal domain of nNOS. Then the nuclear translocation of NF-kappa B was assayed by electrophoretic mobility shift assay (EMSA). The percentage of sub-G1/G0 was determined by flow cytometry (FCM) with propidium iodide (PI).Results1 In these three pain models; SNI was not only the model performed most easily, but also the most sensitive to mechanical stimulation. There is no significant difference in activation among neurons, astrocytes and microglias in the ipsilateral dorsal horn among these three pain models. So SNI model was selected in this study.2 In spinal cord of rats with neuropathic pain, MEK-ERK signaling pathway is activated in astrocytes and so do NF-kappa B in neurons, which may contribute to developments of neuropathic pain.3 The expressions of iNOS and nNOS were increased in RNA level in rat spinal cord following neuropathic pain. The activity of constitutive NOS increased more significantly than inducible NOS (iNOS) in vitro in rat spinal cord following neuropathic pain.4 In dorsal horn of rats with SNI pain model, the high expression of NR2B and nNOS are present, but the transcription of PSD95 in spinal cord was decreased, the interaction between NR2B and nNOS may be disturbed.5 There are at least two kinds of nNOS proteins (weighted at 155 and 135 KDa, e.g. nNOS α and nNOS β ) expressed in rat spinal cord. The expression of nNOS α increased in spinal cord in rats with naturopathic pain, but nNOS β decreased.6 nNOS α and nNOS β have effects on nuclear translocation of NF-kappa B in PC12 cells conversely.ConclusionIt is proved that constitute NOS and inducible NOS play different effects on cell or tissue by different concentration of nitric oxide. In this study, it is indicate that nNOS α from cell membrane and nNOS β in cytoplasm regulate the ability of nuclear factor kappa B conversely. So it may be concluded that nitric oxide can regulate cell function not only in different concentration but also from different place in cell.It is demonstrated that high activity of NF-kappa B is responsible to central sensitization by increasing expression of COX-2 and nNOS in spinal cord of rats with neuropathic pain. In this study, nNOS α which increase the activity of NF-kappa B is found to be increased in spinal cord of rats with neuropathic pain, but expression of nNOS β which decrease the activity of NF-kappa B decrease. Therefore, the expression profile of nNOS may contribute to development of neuropathic pain through regulation of NF-kappa B activity.nNOS α and nNOS β can regulate the ability of nuclear factor kappa B conversely. So those drugs which can affect the activity of nNOS maybe target on nNOS α or nNOS β , then have converse effect on the activity of NF-kappa B. This diversity of neural nitric oxide synthase may explain the controversial effects of nitric oxide in treatment of neuropathic pain.