The data presented herein demonstrate a key role for microglial TLR4 in the induction and maintenance of behavioral hypersensitivity in rodent models of bone cancer pain. Our data also shows that transcription of the microglial activation markers, CD11b and CD14, was significantly elevated at the initiation phase of behavioral hypersensitivity, and moderately increased at the maintenance phase. These results suggest that microglia are involved in the induction and maintenance of behavioral hypersensitivity in rodent models of bone cancer pain, and are not just involved in the initiating phase, as in neuropathic pain and inflammatory pain [19, 20].
The present study successfully established a female rat model of bone pain from metastatic bone cancer. What is in some ways unique about bone cancer pain is that the inflammation, tumor-released products, and tumor-induced injury to primary afferent neurons can simultaneously drive the chronic pain state . There is a prominent up-regulation of glial cells in the spinal cord, ipsilateral to bone cancer pain, and a growing body of evidence suggests that glial cells in the spinal cord play an important role in pain facilitation . A recent report shows that a proliferative burst of glial cells occurs in the ipsilateral dorsal horn following peripheral nerve injury, and that the majority of dividing cells were microglia . The time course of microglia proliferation closely correlated with the development of neuropathic pain, suggesting an important link between microglia activation and pathogenesis of pain hypersensitivity. Although the signals that induce microglial proliferation in response to nerve injury remain incompletely clarified, it has been recently reported that, among the different receptors expressed by microglia, the TLRs, specifically TLR2 and TLR4, are a likely route for microglia activation after nerve injury and play a pivotal role in driving pain hypersensitivity. Our data showed that the spinal microglial activation markers mRNA, TLR4 mRNA and TLR4 protein expression were significantly elevated in the rat model of bone cancer pain. Meanwhile, a report revealed that TLR4-deficient mice have reduced bone destruction following mixed anaerobic infection , which suggests that TLR4 is involved in bone cancer-induced pain perception. In a model of neuropathic pain, spinal microglia TLR4 mRNA expression is also increased after L5 nerve transection in rats . TLR4-knockout and point mutant mice developed less neuropathic pain, showed reduced glial activation, and strongly decreased expression of pain related cytokines . These data shed light on the mechanistic link between microglial TLR4 activation and behavioral hypersensitivity.
This is further evidenced by the present study's finding that functional knockdown of the TLR4 gene by RNAi significantly inhibited bone cancer-induced behavioral hypersensitivity. Consistent with the behavioral test, the real-time RT-PCR study and western-blot analysis demonstrated that TLR4 significantly suppresses spinal TLR4 mRNA and protein expression during bone cancer pain. These data indicate that TLR4 is involved in the spinal transmission and processing of noxious inputs from the peripheral cancer area and facilitates bone cancer hyperalgesia. In the model of neuropathic pain, TLR4 antisense oligonucleotide treatment can significantly attenuate the behavioral hypersensitivity . Repeated administration of a potent TLR4 antagonist (FP-1) could also relieve thermal hyperalgesia and mechanical allodynia in mice with painful neuropathy . These data indicate that TLR4 is not only involved in neuropathic pain, but also in bone cancer pain.
It should also be noted that our research shows that functional knockdown of TLR4 also reduced spinal microglial activation, and reduced the expression of mRNA for spinal proinflammatory cytokines. A previous study demonstrated that IL-1β was upregulated in a bone cancer pain rat model and that intrathecal IL-1ra produced an anti-pain effect in such a model [13, 25]. The CNS innate immune response includes rapid activation of immune effectors cells and the release of proinflammatory cytokines, such as TNF-α, IL-1 β, IL-6, and IFN-β, through the activation of TLR4-MyD88-dependent or -independent pathways . Furthermore, the expression of proinflammatory cytokines by activated glia after the injection cancer cells into the tibia has been shown to be a major factor contributing to the establishment of behavioral hypersensitivity . As shown in the present study, the knockdown of TLR4 expression leads to attenuation of behavioral hypersensitivity, decreased glial activation, and decreased expression of proinflammatory cytokines. Thus, it is very important that TLR4 provides a mechanistic link between microglial activation, innate immunity, and the initiation of behavioral hypersensitivity in the rat model of bone cancer pain.
Like many chronic pain states, bone cancer pain becomes more severe with disease progression, requiring higher doses of analgesics to control the pain. In the present report, we show that intrathecal TLR4 siRNA could prevent bone cancer-induced tactile allodynia and spontaneous pain at an early stage of tumor growth. However, at the late stage, intrathecal TLR4 siRNA can only attenuate, but not completely block, well-established bone cancer pain. This finding suggests that TLR4 is the main mediator in the induction of bone cancer pain, and that there is a potential role for other receptors to be involved in maintaining the pain state [29, 30]. These might include bradykinin, P2X3, TRPV1, and prostaglandin receptors, acid-sensing ion channel 3 and voltage-gated sodium channels, and unique glial/neuronal signals like fractalkine. Thus, our results underscore the complexity of CNS cascades and mediators that may underlie neuronal sensitization, the pathological manifestation of cancer pain.
In vivo delivery of siRNAs into the central nervous system is complicated by the fact that oligonucleotides do not efficiently cross the blood-brain barrier. Recently, chemical modifications of siRNAs have become essential for achieving high levels of gene silencing, enhancing plasma stability, and increasing in vivo potency, combined with a low degree of undesired effects . The present study applied RNAi technology that was improved in two ways: (i) chemical modification of the siRNA. The introduction of certain constituents, such as fluorine, into the ribose 2' position of the primers renders siRNAs more resistant to nuclear acid enzymes. In addition, the 3'-end of the siRNA antisense strand has the special role of identification of the target mRNA, and chemical modification at the 3'-end of the antisense strand will lead to a significant increase in interference. Therefore, we modified our siRNAs with one 2'-FU substitution at the 3'-end of the sense strand, but left the antisense chain unmodified, by which we hoped for enhanced nuclease resistance combined with good specificity. (ii) Application of an appropriate delivery system. We applied in vivo jetPEI™ (polyethyleneimine, PEI) as the siRNA delivery system. PEI is a cationic polymer nanoparticle that, compared with liposomes or viral vector, can decrease cytotoxicity, and can avoid potential vector immunogenicity and tumorigenicity, when used as an siRNA delivery system. Growing evidence indicates that PEI-siRNA is an ideal tool for inhibiting specific gene expression .