Role of the blood-spinal cord barrier: An adheren junction regulation mechanism that promotes chronic postsurgical pain.

Chronic postsurgical pain (CPSP) is a serious postoperative complication with high incidence, and its pathogenesis involves neuroimmune interactions and the breakdown of the blood-spinal cord barrier (BSCB), the decreased level of adheren junction (AJ)-related proteins is an important cause of BSCB injury. Vascular endothelial-cadherin (VE-cadherin) and p120 catenin (p120) constitute the endothelial barrier adheren junction. The Src/p120/VE-cadherin pathway is involved in the regulation of the endothelial barrier function. However, the role of the BSCB-AJ regulatory mechanism in CPSP has not been reported. In this study, we established a skin/muscle incision and retraction (SMIR) model and evaluated the paw withdrawal threshold (PWT), the effects of an Src inhibitor and p120 knockdown on p-Src, p120 and VE-cadherin expression, as well as BSCB-AJ function in rat spinal cord were observed to explore the regulation of BSCB-AJ function by the p-Src/p120/VE-cadherin pathway in promoting SMIR-induced CPSP. The levels of p-Src, p120 and VE-cadherin in the spinal cord were detected by Western blot. Meanwhile, BSCB permeability test was used to detect the changes in BCSB function. Finally, the spatial and temporal localization of p120 in spinal cord was detected by immunofluorescence. Our findings indicated that p-Src/p120/VE-cadherin could induce BSCB-AJ dysfunction and promote the development of CPSP.

CAV-1 Overexpression Exacerbates the Manifestation in EPAC-1-Induced Chronic Postsurgical Pain in Rats.

Purpose: Caveolae (CAV) are an invaginated microcapsule with the shape of Ω on the surface of the cell membrane. Caveolin-1 (CAV-1) is involved in neuropathic pain, and adenosine monophosphate (AMP)-exchange protein directly activated by cAMP1 (EPAC-1) is a potential therapeutic target for chronic pain. However, whether EPAC-1 promotes chronic postsurgical pain (CPSP) through CAV-1 has not been reported. Here, we aim to investigate the underlying mechanism of CAV in CPSP. Methods: All the rats were divided into 9 groups, including the Naive group, Sham group, skin/muscle incision and retraction (SMIR) group, SMIR + CAV-1 siRNA group, SMIR + control siRNA group, SMIR (7 days)+Saline group, SMIR (7 days)+CE3F4 group, 8-PCPT group, and Saline group. The CPSP rat model was established after SMIR. A mechanical withdrawal threshold (MWT) was recorded to evaluate the animal's behavior. Western blotting and immunofluorescent were performed to detect the protein expression levels of EPAC-1 and P-CAV-1. Results: EPAC-1 and CAV-1 were both overexpressed after operation, particularly in astrocytes, microglia, and neurons of spinal marrow (all P < 0.05). Interestingly, CAV-1 siRNA can partly reverse the SMIR-induced hypersensitivity, but there was no effect on EPAC-1. Besides, EPAC-1 blockage partly reversed the SMIR-induced hypersensitivity and CAV-1 overexpression, and EPAC-1 activation promoted CAV-1 overexpression and hypersensitivity in normal rats (all P < 0.05). Conclusion: CAV-1 mediates the functional coupling of microglia, astrocytes, and neurons, and thus EPAC-1/CAV-1 plays an important role in CPSP exacerbation.

Role of caveolin-1 in chronic postsurgical pain in rats.

Chronic postsurgical pain (CPSP) has a high incidence, but the underlying mechanisms remain elusive. Previous studies have indicated that caveolin-1 (Cav-1) plays a notable role in pain modulation. To study the role of Cav-1 in CPSP in the present study, a rat model of skin/muscle incision and retraction (SMIR) was established. Under anesthesia, skin and superficial muscle of the medial thigh were incised and a small pair of retractors inserted. It was revealed that SMIR increased the expression of Cav-1 in the dorsal root ganglion (DRG) and the injured tissue around the incision. Furthermore, the infiltration of endothelial cells and macrophages in the injured tissue around the incision increased constantly, and the vascular permeability increased due to the destruction of the vascular endothelial barrier function around the injured tissue. Cav-1 was mainly expressed by CD68-positive macrophages and CD34-positive endothelial cells in the injured tissues around the incision, while it was also primarily localized in the medium and large neurofilament 200-positive neurons and a small number of calcitonin gene-related peptide- and isolectin B4-positive small and medium-sized neurons in the DRG. The results demonstrated that the sustained high expression levels of Cav-1 in the injured tissue around the incision could lead to the dysfunction of the vascular endothelial barrier and, thus, could induce the inflammatory response through the lipoprotein transport of endothelial cells, thereby resulting in peripheral sensitization. In addition, the sustained high expression levels of Cav-1 in the DRG could sensitize large-sized neurons and change the transmission mode of noxious stimuli. The findings of the present study indicated that a Cav-1-mediated process could participate in neuronal transmission pathways associated with pain modulation.

Role of p120 Catenin in Epac1-Induced Chronic Postsurgical Pain in Rats.

Chronic postsurgical pain (CPSP) is a chronic pain state that is difficult to be treated clinically. A series of complicated changes have been produced from nociceptive stimulation to the occurrence and development of postsurgical pain. Many mechanisms remain unclear. In order to study the role of intercellular gap junctions in inducing inflammatory microenvironment at the beginning of nociceptor after operation, the model of skin/muscle incision and retraction (SMIR) was established. We observed the changes of the expression of exchange proteins directly activated by cAMP-1 (Epac1) and p120 catenin (p120), the quantities of macrophages and endothelial cells, vascular endothelial permeability, and mechanical withdrawal threshold (MWT). It was found that macrophages and endothelial cells were functionally coupled through Epac1-p120. Adhesive linkage disorder remodeled the chronic, inflammatory, and eutrophic microenvironment at the beginning of nociceptor after operation through macrophages, endothelial cells, and endothelial paracellular pathways. It might be an early event and a key step in peripheral sensitization of CPSP. The expression of p120 in muscle tissue around the incision might become a prognostic marker for the conversion of acute postsurgical pain into CPSP. Targeted intervention of Epac1-p120 might be a clinical strategy for inhibiting the conversion of acute postsurgical pain into CPSP.

PKCßII-induced upregulation of PGP9.5 and VEGF in postoperative persistent pain in rats.

PURPOSE: Postoperative pain is a common clinical problem. In this study, we aimed to investigate the role of protein kinase C ßII (PKCßII) in the progression of postoperative pain following skin/muscle incision and retraction (SMIR) surgery. MATERIALS AND METHODS: SMIR postoperative pain model was established in rats, akin to a clinical procedure. The expression level and location of p-PKCßII were observed in dorsal root ganglion (DRG) or spinal cord from SMIR-operated rats by Western blotting and immunofluorescence. In addition, the effects of PKCßII on the expression of protein gene product 9.5 (PGP9.5) or vascular endothelial growth factor (VEGF) were assessed by using pharmacological activator and inhibitor of PKCßII. Moreover, mechanical withdrawal threshold (MWT) was assessed before or after SMIR-operated rats were treated with inhibitor or activator of PKCßII. RESULTS: The expression of PKCßII in DRG and spinal cord was significantly increased after SMIR surgery (P < 0.001, P < 0.01) and expression of PKCßII was located in the neurons of the spinal cord, and magnocellular neurons, non-peptide neurons, and peptide neurons in DRG. Besides, compared with skin/muscle incision group, retraction caused a marked increase in the expression of PKCßII and a significant decrease of MWT (P < 0.001, P < 0.05). The activator of PKCßII greatly increased the expression of PGP9.5 and VEGF (P < 0.05, P < 0.01) and enhanced MWT (P < 0.001), while inhibitor of PKCßII decreased the expression of PGP9.5 and VEGF and attenuated MWT (P < 0.05, P < 0.01, P < 0.001). CONCLUSION: Activation of PKCßII signaling pathways might be an important mechanism in the progression of postoperative pain.

Peripheral KATP activation inhibits pain sensitization induced by skin/muscle incision and retraction via the nuclear factor-κB/c-Jun N-terminal kinase signaling pathway.

The aim of the current study was to assess the effect of pinacidil activation of ATP­sensitive potassium (KATP) channels prior to skin/muscle incision and retraction (SMIR) surgery on peripheral and central sensitization, and investigate molecular interferential targets for preventive analgesia. Male Sprague-Dawley rats were randomly assigned to one of the following five groups: Control, incision (sham surgery), incision plus retraction (SMIR) group, SMIR plus pinacidil (pinacidil) group and the SMIR plus pyrrolidine dithiocarbamate (PDTC) group. The rats in the pinacidil and PDTC groups were intraperitoneally injected with pinacidil or PDTC, respectively, prior to the SMIR procedure. The mechanical withdrawal threshold (MWT) was determined. Western blotting was performed to detect the alterations in the subunits of the KATP channels, Kir6.1 and SUR2, levels of nuclear factor­κB (NF­κB) in the tissue around the incision and c­Jun N­terminal kinase (JNK) in the spinal cord. There was a significant increase observed in the levels of NF­κB and JNK following SMIR surgery compared with the control group, and a significant reduction in MWT and the levels of Kir6.1 and SUR2. Additionally, intraperitoneal administration of pinacidil inhibited the reduction in MWT, and Kir6.1 and SUR2 levels. SMIR was observed to result in increases in the levels of NF­κB and JNK. In addition, in the PDTC group, the alterations in MWT, NF­κB, JNK, Kir6.1 and SUR2 resulting from SMIR were blocked. The results of the current study suggest that the deteriorations in the microenvironment resulting from the SMIR procedure can induce peripheral and central sensitization, and that the activation of peripheral KATP by pinacidil prior to SMIR is able to inhibit peripheral and central sensitization via the NF­κB/JNK signaling pathway, thus resulting in preventive analgesia.

Effect of Epac1 on pERK and VEGF Activation in Postoperative Persistent Pain in Rats.

The function of guanine nucleotide exchange protein conversion factor (Epac1) in regenerating nerves, stimulating insulin release, controlling vascular pressure, and controlling other metabolic activities has been recognized; however, few studies have addressed the potential role of Epac1 in controlling chronic postoperative pain. Using a rat model of skin/muscle incision and retraction (SMIR), our study tested the hypothesis that increased Epac1 signaling is a factor in postoperative chronic pain. Rats were randomly divided into normal, sham operation, SMIR, SMIR + Epac1 siRNA (Epac1 inhibitor), and normal + 8-pCPT (Epac1 agonist) groups. The mechanical withdrawal threshold (MWT) was used as an index of pain sensitivity. Epac1 expression in the incision-site muscle, DRG, and spinal cord was assessed using western blotting and immunofluorescence. The effects of Epac1 agonists and Epac1 siRNA on MWT and phosphorylated extracellular signal-regulated kinase (pERK), vascular endothelial growth factor (VEGF), protein release in the spinal cord, and DRG levels were also studied. SMIR increased Epac1 expression in the incision-site muscle, spinal cord, and DRG, and decreased MWT. 8-pCPT induced the development of hypersensitivity and increased pERK and VEGF expression in normal rats, whereas siRNA decreased the expression of pERK and VEGF. This study suggests that activation of the Epac1 signal might induce local postoperative recovery process, which could be an important mechanism by which to control postoperative chronic pain. Our data suggest that therapy targeted at decreasing Epac1 levels provides promise for the prevention and treatment of chronic postoperative pain.

ATP-sensitive potassium channels alleviate postoperative pain through JNK-dependent MCP-1 expression in spinal cord.

Although adenosine triphosphate-sensitive potassium (KATP) channels have been proven to be involved in regulating postoperative pain, the underlying mechanism remains to be investigated. In this study, we aimed to determine the role of spinal KATP channels in the control of mechanical hypersensitivity in a rat pain model, in which rats were subjected to skin/muscle incision and retraction (SMIR) surgery, as well as in LPS-stimulated astrocytes. The results showed that KATP channel subunits Kir6.1, SUR1 and SUR2 were normally expressed in the spinal cord and significantly downregulated after SMIR. SMIR caused a marked increase in monocyte chemoattractant protein-1 (MCP-1) mRNA expression and in the protein level of p-JNK in the spinal cord. Intrathecal administration of a KATP channel opener pinacidil (Pina) suppressed mechanical allodynia after SMIR and significantly downregulated the MCP-1 mRNA expression and the protein level of p-JNK induced by SMIR. Inverted fluorescence microscopy showed that Kir6.1 was co-localized with astrocytes only and SUR2 was co-localized primarily with neurons, in a small amount with astrocytes. Furthermore, in vitro studies showed that following incubation with LPS, the astrocytic MCP-1 mRNA expression and p-JNK content were markedly increased, whereas the mRNA levels of Kir6.1 and SUR2 were significantly downregulated in astrocytes. KATP channel opener pinacidil inhibited the LPS-triggered MCP-1 and p-JNK elevation in rat primary astrocytes. The results suggested that KATP channel opener treatment is an effective therapy for postoperative pain in animals, through the activation of the JNK/MCP-1 pathway in astrocytes.

Effect of adenosine triphosphate-sensitive potassium activation on peripheral and central pain sensitization.

BACKGROUND: Alterations in adenosine triphosphate-sensitive potassium (KATP) activity and expression under changing physiological conditions are important adaptive and protective mechanisms. KATP subunit expression is also altered in neuropathic pain; whether these changes are adaptive or deleterious is unclear. We therefore established a skin/muscle incision and retraction (SMIR) rat model of postoperative pain and examined the relationship between pain sensitization and changes in KATP subunit expression. METHODS: Rats were randomly divided into untreated, sham-operation, SMIR, and SMIR + Pinacidil (sulfonylurea receptor [SUR]2-activator) groups. In the SMIR group, skin and muscle were retracted for 1 h after incision. In the SMIR + Pinacidil group, Pinacidil was injected intraperitoneally 0.5 h before SMIR or into the spinal myelin sheath 7 d after SMIR. Mechanical withdrawal threshold was used as an index of pain sensitivity. Expression levels and localization of the KATP subunits Kir6.2, Kir6.1, SUR1, and SUR2 were measured by Western blotting and immunofluorescence. RESULTS: A rat postoperative pain model was successfully established, in which SMIR induced mechanical hypersensitivity (allodynia). Notably, significantly increased Kir6.1, Kir6.2, SUR1, and SUR2 protein expression levels were observed in tissues around the incision (P < 0.05). In addition, significantly decreased Kir6.1, SUR2, and SUR1 protein levels were obtained in spinal cord L3-L5. SMIR also starkly increased nerve growth factor expression in the muscle around the incision. Importantly, intrathecal Pinacidil injection inhibited the overexpression of allodynia markers and nerve growth factor. CONCLUSIONS: Hyperexcitability due to spinal Kir6.1 and SUR2 downregulation may be responsible for postoperative pain. SUR2 activation is a potential strategy to inhibit postoperative allodynia.

Effects of pinacidil on changes to the microenvironment around the incision site, of a skin/muscle incision and retraction, in a rat model of postoperative pain.

The aim of the present study was to evaluate the influence of the microenvironment around an incision site, on peripheral and central sensitization. The effects of pinacidil activation of ATP-sensitive potassium (KATP) channels prior to skin/muscle incision and retraction (SMIR) surgery were assessed. A total of 24 male Sprague Dawley rats were randomly assigned to four groups: Control, sham (incision operation), SMIR (incision plus retraction 1 h after the skin/muscle incision) and pinacidil (SMIR plus pinacidil). The rats in the pinacidil group were intraperitoneally injected with pinacidil prior to the SMIR procedure. The mechanical withdrawal threshold (MWT) was determined at each time point. The microvessel density (MVD) value was determined by immunohistochemistry, and western blotting was performed to analyze the relative protein expression levels of nerve growth factor (NGF), glucose transporter protein-1 (GLUT1) and C-jun N-terminal kinases. There was a significant reduction in the levels of MVD, GLUT1 and MWT following SMIR surgery as compared with the incision alone, and a significant increase in the NGF protein expression levels. In the SMIR group, the MVD value was significantly increased seven days after surgery, as compared with three days after surgery. Additionally, intraperitoneal administration of pinacidil prior to the SMIR surgery inhibited the SMIR­induced reduction in MWT and MVD and attenuated the SMIR­induced GLUT1 reduction. The results of the present study suggest that the microenvironment around an incision site may affect the development of peripheral and central sensitization. In addition, pinacidil had an inhibitory effect on the formation of the inflammatory microenvironment around the incision site through activation of KATP channels, thereby inhibiting peripheral and central sensitization.

[A study on cytokine levels and nitric oxide content in rabbit aqueous humor after lens extraction and intraocular lens implantation].

OBJECTIVE: To investigate the relationship among inflammatory reaction and cytokine levels, nitric oxide (NO) content in aqueous humor after intraocular lens implantation. METHODS: Eighteen New Zealand rabbits were divided randomly into 3 groups (each 6 rabbits): (1) control group, (2) extracapsular cataract extraction group (ECCE) and (3) ECCE and posterior chamber intraocular lens implantation group (ECCE + IOL). The inflammation of all experimental rabbit eyes were observed by a zoom-photo slit-lamp microscope 0, 1, 3, 7, 14, 30 days postoperatively, including corneal edema and anterior chamber exudation. Meanwhile, aqueous humor was drawn for white blood cell (WBC) count and classification, as well as for NO(2)(-)/NO(3)(-) and cytokine assays, including interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha). Statistics were taken by SPSS software. RESULTS: (1) The anterior chamber exudation was the most serious and monocyte/macrophage in aqueous humor were the highest in ECCE + IOL group in postoperative 7 - 14 days. (2) The levels of IL-2, TNF-alpha and the content of NO(2)(-)/NO(3)(-) in aqueous humor of ECCE + IOL group were higher than that in ECCE group and control group in the postoperative 1 - 14 days respectively, and they increased to their peak values at the postoperative 3 - 7 days and decreased gradually after postoperative two weeks. (3) The change regularity of IL-2, TNF-alpha, NO(2)(-)/NO(3)(-) and inflammatory reaction in each group were basically similar, i.e. the more serious the reaction, the higher the levels of the contents. CONCLUSION: The intraocular inflammation after intraocular lens implantation is closely related to the changes of cytokine levels and NO content in aqueous humor.