Th17 Cells Infiltration Into Spinal Cord Of SNL Rat And Possible Central Sensitization Mechanism

Neuropathic pain refers to "the pain initiated or caused by a primary lesion or dysfunction of the nervous system". It may be induced by infection, nerve trauma, metobolic disease, ischemia, radiotherapy or chemotherapy, et al. Central sensitiztion has been applied to explain neuropathic pain, though its mechanism has not been well known. Central sensitization in the spinal cord is well be emphasized at present, whereas it is also produced in the thalumas and brain cortex. Intensive damage can cause activity-dependent central sensitization, however, central sensitization of neuropathic pain is mostly led by glia cells activation, peripheral immune cells infiltration and chronic nerve inflammation.It has been demonstrated that Th1, Th17 cells infiltration into injured nerve was involved peripheral sensitization, but Th2 cells could attenuate pain sensitivity. Recently, CD4+T cells infiltration in spinal cord was also found to promote neuropathic development. Infiltrated Th17 cells in CNS has been shown to be involved in autoimmune diseases with neuropathic pain, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). To further understand the role of immune cells in NP, the sub-types of CD4+T cells infiltrated in spinal cord after nerve injury were identified in vivo and in vitro; as well as to study the possible mechanism that Th17 cells are involved in central sensitization? Objective(1) CD4+T cells migrated and infiltrated into spinal cord after nerve injury, our study aims to clarify the specific sub-type of infiltrated CD4+T cells in spinal cord and its molecular mechanism.(2) It is well known that microglias and astrocytes played an important role in neuropathic pain, the former contributed to the initiation of neuropathic pain, and the latter to the maintenance. We found CD4+Th17 cells infiltrated in spinal cord during the maintenance phase of neuropathic pain. This experiment explores the relationship between infiltrated Th17 cells and activated astrocytes and its central sensitization mechanism.(3) Among the elders, postherpetic neuralgia is a common disease of neuropathic pain. The present work was to investigate the proportion of peripheral T lymphocytes and the associated inflammatroy cytokines expression in PHN. To understand the possible mechanism of the central sensitization effects on neuropathic pain, the intervention through anti-inflammation and anti-nociceptive transmission were taken.Methods(1) Establishment and identification of the rat model of spinal nerve ligationMale adult SD rats were anesthetized using pentobarbital sodium, a longitudinal incision overlying the L4-S1 was made and the left muscle tissue was seperated, The L6 transverse process, L6/S1 posterior interarticular process was exposed and transected. Carefully tease the tissue and expose L5 and L6 spinal nerve, the distal nerve of L5 and L6 were gently separated, firmly ligated with 5-0 silk suture and transected. The wound was then inspected for homostasis, washed and closed. NS was administered i.p. after surgery, and the animal was allowed to emerge from anesthesia in the warming place. In the case of sham-operated animals, an identical exposure was performed, but the nerve was not ligated.According to the experimental procedure, mechanical sensitivity of the rats' left and/or right feet was measured at days 1,3,5,7,10,14,21 after surgury using Up and Down methods.50% mechanical withdrawl threshold (50%MWT) was calculated. If the value of 50%MWT exceeded 4.0 g at days 7 post sugury, the animal was considered failed preparation.(2) FACS detecting the infiltrated CD4+T cells in spinal cordThe rat's heart was exposed and perfused with NS. L4-L6 of spinal cord was separated and harvested. Each FACS sample consisted of three animals. Mononuclear cells from spinal cord tissue were obtained with discontinuous Percoll gradients methods, then they were suspended in 100μL PBS containing anti-rat-CD3-FITC and anti-rat-CD4-PE and incubated at 4 degrees for 30 minutes. After that, the cells were washed and fixed with 4% formaldehyde for whole night. Next day, analysis was performed through FACS.(3) Analysis of the infiltrated Th17 cells in spinal dorsal hornThe rats were transcardially perfused with NS followed by 4% formaldehyde. L5-L6 segment of lumbar spinal cord was harvested, fixed in 4% formaldehyde and dehydrated in 30% sucrose at 4 degrees. Tissue was then freeze-mounted in OCT embedding medium on cork blocks for cryostat sectioning. Fluorescent immunohistochemistry for CD4 and IL-17 was conducted. The left fresh spinal dorsal horn from L4-L6 segment was harvested, RT-qPCR determined the mRNAs expression of IL-17, RORγt, CCL2, CCL20, CXCL10 mRNA. ELISA analyzed the serum level of IL-17, IFNγ, IL-4 according to the product instruction. Accoding to our results, Th17 infiltration in spinal cord and its molecular mechanism were determined.(4) Exploring central sensitization mechanism of Th17 cellsIn vivo, Fluorescent immunohistochemistry detected GFAP of L5-L6 spinal cord, RT-qPCR measured the mRNAs expression of imflammatory cytokines of IL-1β,TNFα, IL-6. In vitro, different concentration of IL-17 stimulated astrocytes. Following stimulation, MTT investigated the astrocytes proliferation, RT-qPCR measured the mRNAs expression of IL-1β, IL-6 and TNF-αin astrocytes. Consquently, central sensitization mechanism of Th17 cells was concluded. (5) Immune function assessment of PHN patients and central interventionFACS analyzed sub-type of T cells in blood,ELISA measured serum level of IL-12, IL-17, IFNγ, IL-4, IL-15 and IL-1α. Furthermore, central intervention was performed through epidural diprospan with or without intrathecal midazolam, then VAS score and its adverse efffects were assessed.Results(1) Twenty rats were performed to nerve ligation, two rats were damaged the L4 nerve at the initial experimental stage, two showed 50%MWT more than 4.0 g, the four rats were excluded from further analysis. The paw on the operative side presented moderate eversion and the toes were held together. Abnormal posture and spontaneuous pain also were found in nerve ligated rats. At days 3 post nerve ligation surgury, 50%MWT decreased significantly (P<0.001), and reached the minimum value at days 21 compared with sham and control group (P<0.001); During days 10～14, "Mirror pain" was observed on the right foot after nerve ligation.(2) CD4+T cells infiltrated into spinal cord were only found on day 7 post nerve ligation surgery. They accounted for 10.08% of mononuclear cells. IF determined that they were Th17 cells and located at superficial laminae of spinal dorsal horn. Th17 infiltration was accompanied by mRNA significant up-regulation of IL-17, RORγt, CCL2 and CCL20 (P <0.05, P<0.01), howerer, the chemokine CXCL10 was no significant changes. Whereas, on day 7 and 14, the serum level of IL-17, IFNγand IL-4 were no obvious changes among nerve ligation group, sham, control group, respectively.(3) On day 7, GFAP expression in nerve ligation group increased at superficial laminae of spinal dorsal horn; Morever, the mRNA expression of IL-1β, IL-6 also enhanced significantly comparing with sham and control group in the spinal dorsal horn (P<0.01, P<0.05); On day 14, IL-6 mRNA still increased obviously in nerve ligation group (P<0.05) No significant differences in TNF-αmRNA were determined among three groups on day 7and 14. The astrocytes were isolated from the rat and treated by IL-17. After 3 days, the astrocytes were obvious proliferation.(4) There were no significant differences in the ratio of T cells sub-type between PHN and control group, although PHN group showed a downward trend. Furthermore, the serum cytokines level of IL-17, IFNγ, IL-4, IL-12, IL-15 and IL-1αalso didn't present any significant change yet. Intervention on central sensitization through epidural diprospan with or without midazolan both obtained good analgesic effects in two months, the VAS score decreased significantly at any time point after intervention compared to the basal value (P<0.001), and diprospan combined midazolan treatment gained better analgesia, especilly during 6～8W (P <0.001)Conclusions(1) Ligation of L5, L6 unilateral nerve in rat can prepare successfully animal SNL model of neuropathic pain. The surgical procedure of SNL model is stereotyped, and the preparative success rate is high, mechanical pain sensitivity occurs earlier and obviously. The model well manifests the symptom of human patients with causalgia. Unilateral spinal nerve ligation induced contralateral "mirror pain" phenomena.(2) Th17 cells are the primary infiltrated CD4+T cells on day 7 post surgery, which attributed to high expression chemokines of CCL2 and CCL20.(3) The results of in vivo and in vitro experiments indicated that Th17 cells may be involved in the central sensitization. On the one hand, IL-17 combined with IL-17 receptors on astrocytes membrane, stimulated astrocytes to proliferate and activate; then, nociceptive signal spreaded and were maintained through activated astrocytes; On the other hand, IL-17 promoted astrocytes to secrete inflammatory cytokines, which increased directly or indirectly nociceptive neuron excitabilty, therefore maintained neuropathic pain. This mechanism also explained why the temporal Th17 infiltration had long effect on neuropathic pain.(4) Although, among the olders, PHN often developed after viral infection. Our results indicated that the systemic immune function didn't significantly change. The interaction of nerve and immune in PHN is similar to SNL models, primarily in the local injured nerve and central nerve system.(5) Anti-central sensitization treatment involving in antinflammatory and inhibited transmission of nociceptive signal in central nerve system decreased pain sensitivity significantly, which reversly demonstrated central sensitization played an important role in neuropathic pain. Morever, it also accummulated experimental data to explore the curing strategies of PHN.