Background & Aims
Complement compound 5a (C5a) is implicated in promoting and sustaining neuroinflammation, contributing to the persistence of chronic pain states [1]. It acts via 2 receptors – C5aR1 and C5aR2 [2], which cause cytokine production via glial cells in the central nervous system (CNS) [3] and immune cells in the peripheral nervous system (PNS) [4]. This neuroinflammation process is widespread on all levels of the ascending pain pathway and seems to be time-dependent with the first impact on sensory neurons of dorsal root ganglions (DRGs) and their dendrites [1,5]. The present study aims to investigate if, when and how C5a involved in neuropathic pain chronisation.
Methods
The chronic constriction injury (CCI) model of the left sciatic nerve was used in 4 groups of wild-type (WT) mice. Mice were euthanised on the different days post-surgery: 4, 7, 14, and 28. Mechanical allodynia was measured in all groups by the Von Frey test weekly. Additionally, mice in groups euthanised on days 14 and 28 were assessed by CatWalk system for gait parameters; and mice from groups euthanised on days 4 and 7 were assessed by dry ice test for cold allodynia after CatWalk showed uncertain results. Plasma was collected weekly. At the endpoint, DRGs (L3-L5), spinal cord, and brain were taken for immunohistochemical analysis (IHC). Additionally, 2 groups of mice were euthanised on days 4 and 7 to collect liver and nervous tissues (the same as for IHC) without a fixative agent for multiplex analysis of cytokine production and RT-PCR of C5. Plasma C5a was measured by ELISA, neuroinflammation by ICH for astrocytes and microglia in CNC and satellite glia and macrophages in DRGs.
Results
Mice have developed neuropathic pain syndrome in each group reaching paw withdrawal thresholds (PWT) value of 0.4-0.6 g. The plasma level of C5a in the control group was measured on days 4, 7,13, and 25 after CCI and compared with normal C5a plasma level (non-operated mice). For statistical analysis, the mean?±?SEM of concentration was analysed by one-way ANOVA with Dunnett’s Multiple Comparison test. Data showed that C5a in plasma was growing from day 4 till day 7 and then fluctuated on the same level. There was a significant difference (p<0.01) between all groups with control as well as concentration on day 4 was significantly different from day 7.
Conclusions
The increase of C5a in plasma between days 4 and 7 indicates the possibility of neuroinflammation process development. Interrupting this process holds the potential to exert influence on chronic pain development, suggesting a strategic avenue for intervention in managing this condition.
References
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2.Hall, B.E., et al., Transcriptomic analysis of human sensory neurons in painful diabetic neuropathy reveals inflammation and neuronal loss. Scientific Reports, 2022. 12(1): p. 4729.
3.Griffin, R.S., et al., Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. J Neurosci, 2007. 27(32): p. 8699-708.
4.Quadros, A.U., et al., Complement receptor C5aR1 signaling in sensory neuron-associated macrophages drives neuropathic pain. bioRxiv, 2022: p. 2022.07.01.498487.
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