Li-na Wang

Involvement of CX3CR1 in bone cancer pain through the activation of microglia p38 MAPK pathway in the spinal cord.

Previous studies have demonstrated that fractalkine, a newly discovered chemokine, is implicated in spinal cord neuron-to-microglia activation signaling as well as mediation of neuropathic and inflammatory pain via its sole receptor CX3CR1, which is specifically expressed on microglia. However, whether it is involved in bone cancer pain (BCP) and the underlying mechanisms have not been elucidated. In this study we utilized a Sprague-Dawley rat animal model, and our findings indicated that on day 6, 12, and 18 following bone cancer pain induced by Walker 256 cell inoculation, the expression level of CX3CR1 in the spinal cord gradually increased. Intrathecal injection of a neutralizing antibody against CX3CR1 not only delayed the initiation of mechanical allodynia, but also attenuated established pain sensitization of BCP rats. Furthermore, we demonstrated that blockade of CX3CR1 suppressed the activation of microglia and the expression of p38 mitogen-activated protein kinase (MAPK) in the spinal cord in BCP rats. These results suggest a new mechanism of BCP, in which the microglia CX3CR1/p38 signaling cascade potentially plays an important role in facilitating pain processing in BCP rats.

Involvement of spinal monocyte chemoattractant protein-1 (MCP-1) in cancer-induced bone pain in rats

In this study, we examined the involvement of chemokine monocyte chemoattractant protein-1 (MCP-1) in the spinal cord of a rat model of cancer-induced bone pain (CIBP). In this model, CIBP was established by an intramedullary injection of Walker 256 cells into the tibia of rats. We observed a significant increase in expression levels of MCP-1 and its receptor CCR2 in the spinal cord of CIBP rats. Furthermore, the intrathecal administration of an anti-MCP-1 neutralizing antibody attenuated the mechanical allodynia established in CIBP rats. Likewise, an intrathecal injection of exogenous recombinant MCP-1 induced a striking mechanical allodynia in naïve rats. These results suggest that increases in spinal MCP-1 and CCR2 expression are involved in the development of mechanical allodynia associated with bone cancer rats.

Brain-derived neurotrophic factor modulates N-methyl-D-aspartate receptor activation in a rat model of cancer-induced bone pain.

Brain‐derived neurotrophic factor (BDNF) released within the spinal cord induces phosphorylation of N‐methyl‐D‐aspartate (NMDA) receptors on the spinal cord neurons. This process is necessary for maintaining pain hypersensitivity after nerve injury. However, little is known about the role of BDNF and NMDA receptors in cancer‐induced bone pain (CIBP), whose features are unique. This study demonstrates a critical role of the BDNF‐modulated NMDA subunit 1 (NR1) in the induction and maintenance of behavioral hypersensitivity in a rat model of CIBP, both in the spinal cord and in the dorsal root ganglia (DRG). We selectively suppressed BDNF expression by RNA interference (RNAi) using intrathecal administration of BDNF small interfering RNA (siRNA). Then, we assessed mechanical threshold and spontaneous pain in CIBP rats. Real‐time PCR, Western blotting, and fluorescent immunohistochemical staining were used to detect BDNF or NR1 both in vivo and in vitro. BDNF and phospho‐NR1 were expressed under CIBP experimental conditions, with expression levels peaking at day 6 (BDNF) or 9 (NR1). Intrathecal BDNF siRNA prevented CIBP at an early stage of tumor growth (days 4–6). However, at later stages (days 10–12), intrathecal BDNF siRNA only attenuated, but did not completely block, the established CIBP. BDNF‐induced NMDA receptor activation in the spinal cord or DRG leads to central sensitization and behavioral hypersensitivity. Thus, BDNF might provide a targeting opportunity for alleviating CIBP.

Activation of spinal TDAG8 and its downstream PKA signaling pathway contribute to bone cancer pain in rats

Bone cancer pain is difficult to treat and has a strong impact on the quality of life of patients. Few therapies have emerged because the molecular mechanisms underlying bone cancer pain are poorly understood. Recently, T‐cell death‐associated gene 8 (TDAG8) has been shown to participate in complete Freund’s adjuvant‐induced chronic inflammatory pain. In this study, we aimed to examine whether TDAG8 and its downstream protein kinase A (PKA) pathway are involved in bone cancer pain. A bone cancer pain model was made by inoculation of Walker 256 cells into the intramedullary space of rat tibia. Spinal TDAG8 expression was increased after inoculation with tumor cells. Intrathecal TDAG8 siRNA attenuated bone cancer pain behaviors during the initiation and maintenance phases; there were also concomitant decreases in TDAG8 mRNA and protein levels in spinal cord. Moreover, we found spinal PKA and phosphorylated cAMP response element‐binding (pCREB) protein levels were up‐regulated in the rat model of bone cancer pain. Knockdown of TDAG8 resulted in reduced bone cancer pain‐induced spinal PKA and pCREB protein expression in two procedures. Furthermore, intrathecal H‐89 (a PKA inhibitor) significantly attenuated bone cancer pain behaviors in rats. Our results suggest a causal relationship between TDAG8 expression and the initiation and maintenance of bone cancer pain. Activation of spinal TDAG8 contributes to bone cancer pain through the PKA signaling pathway in rats. These findings may lead to novel strategies for the treatment of bone cancer pain.

P2Y1 purinoceptor inhibition reduces extracellular signal-regulated protein kinase 1/2 phosphorylation in spinal cord and dorsal root ganglia: implications for cancer-induced bone pain

It remains unclear as to whether P2Y1 purinergic receptor (P2Y1R) and the molecules that act downstream, such as extracellular signal-regulated protein kinase 1/2 (ERK1/2), are involved in the development of cancer-induced bone pain (CIBP) in vivo. Here, we investigated the role of the P2Y1R in the modulation of CIBP-associated nociception in spinal cord and dorsal root ganglia (DRG). A CIBP model was established by inoculating Walker 256 gland carcinoma cells into the tibia of female rats. Tactile allodynia and spontaneous pain were assessed using von Frey filaments and ambulatory scores. The results showed that both the paw withdrawal latency to tactile allodynia and the ambulatory score to spontaneous pain were significantly different between the CIBP group and the sham group on days 7-9 post-inoculation (P< 0.01). Furthermore, rats in the CIBP group also showed a progressive increase in ambulatory score, which is different from the sham group (P< 0.01). Furthermore, P2Y1R mRNA and phosphorylated ERK1/2 (p-ERK1/2) protein expression levels were increased in the spinal dorsal horn and DRG of the CIBP group relative to the sham group. However, intrathecal injection of the P2Y1R antagonist MRS2179 decreased P2Y1R mRNA and p-ERK1/2 protein expression in the spinal dorsal horn and DRG (P< 0.01). These results provide evidence that the inhibition of P2Y1R-mediated ERK1/2 phosphorylation in the spinal dorsal horn and DRG can attenuate nociception transmission.

MCP-1 stimulates spinal microglia via PI3K/Akt pathway in bone cancer pain

Accumulating evidence suggests that chemokine monocyte chemoattractant protein-1 (MCP-1) is significantly involved in the activation of spinal microglia associated with pathological pain, at the same time that the phosphatidylinositol 3-kinase/Protein Kinase B (PI3K/Akt) pathway localized in spinal microglia is involved in both neuropathic and inflammatory pain. However, whether there is a connection between MCP-1 and the PI3K/Akt pathway and in their underlying mechanisms in bone cancer pain (BCP) has not yet been elucidated. In the current study, we investigated the expression changes of p-Akt in microglia and OX-42 (microglia marker) after being stimulated with MCP-1 in vitro, as well as in a BCP model that was established by an intramedullary injection of mammary gland carcinoma cells(Walker 256 cells) into the tibia of rats. We observed a significant increase in expression levels of p-Akt and OX-42 in microglia as well as in spinal dorsal horns of BCP rats. Furthermore, the intrathecal administration of an anti-MCP-1 neutralizing antibody or PI3K inhibitor LY294002 reduced the expression of p-Akt or OX-42, and LY294002 attenuated the mechanical allodynia of BCP rats. These results suggest that MCP-1 may stimulate spinal microglia via the PI3K/Akt pathway in BCP.

P2X4 receptor in the dorsal horn partially contributes to brain-derived neurotrophic factor oversecretion and toll-like receptor-4 receptor activation associated with bone cancer pain

Previous studies have suggested that the microglial P2X7 purinoceptor is involved in the release of tumor necrosis factor‐α (TNFα) following activation of toll‐like receptor‐4 (TLR4), which is associated with nociceptive behavior. In addition, this progress is evoked by the activation of the P2X4 purinoceptor (P2X4R). Although P2X4R is also localized within spinal microglia in the dorsal horn, little is known about its role in cancer‐induced bone pain (CIBP), which is in some ways unique. With the present rat model of CIBP, we demonstrate a critical role of the microglial P2X4R in the enhanced nociceptive transmission, which is associated with TLR4 activation and secretion of brain‐derived neurotrophic factor (BDNF) and TNFα in the dorsal horn. We assessed mechanical threshold and spontaneous pain of CIBP rats. Moreover, P2X4R small interfering RNA (siRNA) was administered intrathecally, and real‐time PCR, Western blots, immunofluorescence histochemistry, and ELISA were used to detect the expression of P2X4R, TLR4, OX‐42, phosphorylated‐p38 MAPK (p‐p38), BDNF, and TNFα. Compared with controls, intrathecal injection of P2X4R siRNA could prevent nociceptive behavior induced by ATP plus lipopolysaccharide and CIBP and reduce the expression of P2X4R, TLR4, p‐p38, BDNF, and TNFα. In addition, the increase of BDNF protein in rat microglial cells depended on P2X4 receptor signaling, which is partially associated with TLR4 activation. The ability of microglial P2X4R to activate TLR4 in spinal cord leading to behavioral hypersensitivity and oversecretion of BDNF could provide an opportunity for the prevention and treatment of CIBP.

Toll-like receptor-4/p38 MAPK signaling in the dorsal horn contributes to P2X4 receptor activation and BDNF over-secretion in cancer induced bone pain.

Our previous research suggested that the P2X4 receptor (P2X4R) expression in microglia was involved in the activation of toll-like receptor-4 (TLR4) in the dorsal horn in the rat model of cancer induced bone pain (CIBP). In this study, we focused on whether TLR4- mitogen-activated protein kinases, p38 (p38 MAPK) contributes to P2X4R activation and brain-derived neurotrophic factor (BDNF) over-secretion in CIBP. In in vitro experiment, the results showed that BDNF expression evoked by ATP stimulation was dependent on TLR4-p38. In in vivo experiment, the results demonstrated that an intrathecal injection of TLR4 siRNA alleviated nociception induced by lipopolysaccharide (LPS) plus ATP or CIBP with decreased expression of P2X4R, TLR4, BDNF, interleukin-6 (IL-6) and phosphorylated-p38 MAPK (p-p38 MAPK). Moreover, injection with p38MAPK inhibitor SB203580 resulted in an identical pattern compared with treatment with TLR4 siRNA. Our results demonstrate that the activation of TLR4-p38MAPK-P2X4R signaling in microglial possibility plays an important role in the process of nociceptive transmission in CIBP, suggesting new mechanism and potential therapeutic targets for CIBP.

A modified procedure for lumbar intrathecal catheterization in rats

Objectives: Intrathecal catheterization and drug delivery in rats has always been a very important method for neuroscience and pain research. Although the technique has been continually improved since the first report, the experience gained over the years suggested that some defects remained unsolved. On the basis of modification of the standard epidural needle, lumbar needle, and intrathecal tube, we aimed to develop a simple and practical technique for intrathecal catheterization, which was similar to the ‘needle-through-needle technique’ used in combined spinal-epidural (CSE) anesthesia. Methods: For comparison, rats received intrathecal catheterization via either laminectomy at L3-4 (control group) or our modified method (modification group). The operation time, success rate, and the incidence of postoperative complication were recorded. Thermal paw withdrawal latency, mechanical paw withdrawal threshold, Rota-rod, and body weight were measured on pre-operative day 1 and postoperative day 1–3, 5, 7, 14, 21, respectively. Results: Compared with control group, our modified method was more practical to grasp and could bring about higher success rate, firmer catheters immobility, less weight loss, and minimal mortality. There was no difference between the two groups in spinal cord injury, hematoma, location of lumbar enlargement, and lateral location of the catheters tip. The procedure itself did not interfere with behavioral tests and motor coordination. Conclusion: We suggest that the modified method of intrathecal catheterization is well suitable for long-term behavior and pharmacology research on spinal cord.

Effect of triptolide on the regulation of ATP‑binding cassette transporter A1 expression in lipopolysaccharide‑induced acute lung injury of rats

The aim of this study was to investigate the effect of triptolide on ATP‑binding cassette transporter A1 (ABCA1) expression in lipopolysaccharide (LPS)‑induced acute lung injury (ALI) in rats. Thirty male Sprague Dawley rats weighing 200‑250 g were randomly divided into six groups: Normal (N, n=5), Control (C, n=5), LPS (L, n=5), Triptolide 25 µg (TP1, n=5), Triptolide 50 µg (TP2, n=5) and Triptolide 100 µg (TP3, n=5). The N group was not administered anything; the C group was administered 5 ml/kg normal saline intravenously and 7.5 ml/kg 1% dimethylsulfoxide (DMSO) intraperitoneally; the L group was administered 5 mg/kg 0.1% LPS and 1% DMSO; and the TP1, TP2 and TP3 groups were separately injected with 0.1% LPS and 25, 50 or 100 µg/kg triptolide, respectively. All groups had the same liquid‑injection volume. Arterial blood gases, tumor necrosis factor‑α (TNF‑α) and ABCA1 expression and general pathology were examined following the treatments. It was found that increasing the triptolide dose in the TP1‑3 groups resulted in an increase in the expression of ABCA1 mRNA and protein. As compared with the L group, the ABCA1 expression showed a significant increase in TP2 and TP3 groups (P<0.05). In addition, the expression level of TNF‑α was significantly increased in the L and TP1 groups, as compared with that in the N or C groups (P<0.05). Conversely, a marked decrease in TNF‑α expression was detected in the TP2 and TP3 groups, as compared with the L or TP1 groups (P<0.05). In conclusion, this study found that triptolide could promote the expression of ABCA1 mRNA and protein and inhibit other inflammatory factors during LPS‑induced ALI in rats. Regulating the expression of ABCA1 may be one of the protective mechanisms of triptolide. Furthermore, triptolide‑induced increases in ABCA1 expression occurred in a dose‑dependent manner between 25 and 100 µg/kg.