Objective To investigate the role of spinal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in dexmedetomidine-produced antinociceptic effect.Methods In vivo experiment Thirty wild type C57BL/6J mice and 30 HCN1 gene knockout (HCN1-/-) mice, aged 2-3 months, weighing 19-25 g, were randomly divided into 5 groups (n=6 each) using a random number table: control group (group C), and dexmedetomidine 10, 20, 30 and 40 μg/kg groups (Dex10, Dex20,Dex30 and Dex40 groups).In Dex10, Dex20, Dex30 and Dex40 groups, dexmedetomidine 10, 20, 30 and 40 μg/kg were intraperitoneally injected, respectively.The equal volume of normal saline was given in group C.Before dexmedetomidine administration, and at 15, 30, 45, 60, 75, 90, 105 and 120 min after dexmedetomidine administration, tail flick latency to a thermal nociceptive stimulus was measured, and the percentage of the maximum possible effect (MPE％) was calculated.In vitro experiment HCN1 and HCN2 plasmids and green fluorescent plasmids were transfected into HEK293 cells with liposome 2000.At 24-48 h after transfection, HCN1 and HCN2 channel currents were recorded using whole-cell patch clamp technique.HCN channel currents were recorded as baseline value after rupture of membrane.HEK293 cells were perfused with the extracellular fluid containing different concentrations of dexmedetomidine (0.1, 1.0 or 10.0 μmol/L).After the cells were perfused with dexmedetomidine 0.1 μmol/L for 5 min, HCN currents were recorded.The inhibition rate of currents were calculated.After washout, HCN currents were recorded after the cells were perfused with the next concentration for 5 min.The half-maximal activation voltage (V1/2) of HCN channels and the curve slope were recorded.The difference in V1/2 before and after administration (△V1/2) were calculated.Results Compared with group C, MPE％ was significantly increased in Dex10 group-Dex40 group of wild type and HCN1-/-mice (P＜0.05).Compared with Dex30 and Dex40 groups of wild type mice, MPE％ was significantly decreased in Dex30 and Dex40 groups of HCN1-/-mice (P＜0.05).There was no significant difference in MPE％ between Dex10 group and Dex20 group of wild type and HCN1-/-mice (P＞0.05).Compared with the baseline value, the currents and V1/2 of HCN1 and HCN2 channels in HEK293 cells were significantly decreased when the cells were perfused with dexmedetomidine 0.1, 1.0 and 10.0 μmol/L (P＜0.05).Compared with the value when the cells were perfused with dexmedetomidine 0.1 μmol/L, the currents and V1/2 of HCN1 and HCN2 channels in HEK293 cells were significantly decreased, and inhibition rate of currents and △ V1/2were increased when perfused with dexmedetomidine 1.0 and 10.0 μmol/L (P＜0.05).Compared with the value when the cells were perfused with dexmedetomidine 1.0 μmol/L, the currents and V1/2 of HCN1 and HCN2 channels in HEK293 cells were significantly decreased, and inhibition rate of currents and △ V1/2were increased when perfused with dexmedetomidine 10.0 μmol/L (P＜0.05).There was no significant difference in the activation curve slope of HCN1 and HCN2 channel currents in HEK293 cells when the cells were perfused with dexmedetomidine 0.1, 1.0 and 10.0 μmol/L (P＞0.05).Conclusion Dexmedetomidine-produced antinociceptic effect is likely related to the inhibition of spinal HCN channel opening.

Objective To establish neuropathic pain models,explore the effects and mechanisms of dexmedetomidine on neuropathic pain.Methods Wistar rats were randomly divided into four groups (n =9):0.9％ sodium chloride solution CCI group (N),dexmedetomidine CCI group (D),ZD7288 CCI group (Z) and sham-operated group (Sham).Sciatic nerve ligation was performed in group N,D and Z.The sciatic nerve in group Sham was exposured without ligation.7 d after surgery,the rats in group D were intraperitoneal injected with dexmedetomidine (40 μg· kg-1),and the rats in group Z were intraperitoneal injected with ZD7288 (10 mg·kg-1)once a day for 3 d.The same volume of 0.9％ sodium chloride solution was given at the same time in group N.The behavioral test was performed before and 7 d after operation,as well as 3 d after injection treatment.Mechanical allodynia was assessed by paw withdrawal mechanical threshold (PWMT) to von Frey filaments.Thermal hyperalgesia was assessed by paw thermal withdrawal latency (TWL) to radiant heat.Dexmedetomidine block of HCN channels in dorsal root ganglion (DRG) neurons were confirmed by whole-cell recording.Results 7 d after surgery,the PWMT and TWL of rats in group N,D and Z were decreased significantly (P ＜ 0.05).The PWMT and TWL in group Sham were no significant difference before and after operation.Dexmedetomidine significantly increased the levers of PWMT and TWL in group D and Z after treatment for 3 d,and group Z was greater than group D (P ＜ 0.05).Dexmedetomidine (0.1,1,10 μmol· L-1) caused a concentration-dependent decrease in the amplitude of Ih in DRG neurons from (-844.43 ± 386.34) to (-215.99 ± 63.90) pA (P ＜ 0.05),and the inhibition rate of Ih was (11.87 ± 1.80) ％,(35.26 ± 3.65) ％ and (52.02 ± 5.56) ％,respectively(P ＜0.05).Dexmedetomidine produced a dose-related shift to the left of the Ih activation,and a negative shift in V1/2 (P ＜ 0.05).V1/2 shifted from (-86.21 ± 1.68) to (-103.54 ± 2.01) mV (P ＜ 0.05).The slope values were not altered by dexmedetomidine.Conclusion Dexmedetomidine produces a dose-dependently analgesic effect on neuropathic pain after peripheral never injury,which is likely due to the inhibition of Ih and reduction of ectopic spontaneous discharge in DRG neurons.

Mycobacterium (M.) vaccae is a fast-growing species of saprophytic bacteria that is widely distributed. To understand the host immune responses induced by M. vaccae isolated from bovine submaxillary lymph nodes, C57BL/6 mice were infected with reference strain M. vaccae Bacillus Calmette-Guérin (BCG) and isolated M. vaccae using intraperitoneal injections. Comparison of the bacterial replication and organ pathology between M. vaccae and M. vaccae BCG revealed that M. vaccae was more malignant than M. vaccae in mice. We also demonstrated that serum from the M. vaccae-infected mice contained a higher expression level of gamma-interferon (IFN-γ), tumor necrosis factor alpha, monocyte chemoattractant protein-1, interleukin (IL)-4, IL-12, IL-10 and transforming growth factor beta than did the other groups, especially after week 4. Furthermore, when the numbers of CD3⁺CD4⁺IFN-γ⁺ and CD3⁺CD4⁺IL4⁺ cells in the infected mice were observed by flow cytometry, we found that a powerful T helper 1 (Th1) response was induced by M. vaccae infection, which was associated with the emergence of CD3⁺CD4⁺IFN-γ⁺ cells. However, the Th1 response declined over time, which was associated with appearance of the CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD152⁺Treg cell reaction. In addition, a strong Th2 response was found. Finally, we found that M. vaccae infection increased the production of type I IFNs, which was associated with a reduced Th1 response.
Animals ; Bacillus ; Bacteria ; Chemokine CCL2 ; Flow Cytometry ; Injections, Intraperitoneal ; Interferon-gamma ; Interleukin-10 ; Interleukin-12 ; Interleukins ; Lymph Nodes ; Mice* ; Mycobacterium bovis ; Mycobacterium* ; Pathology ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha