Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1 Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Calcium channel α2δ1 proteins mediate trigeminal neuropathic pain states associated with aberrant excitatory synaptogenesis.

To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Thrombospondin-4 contributes to spinal sensitization and neuropathic pain states.

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Application of pulsed radiofrequency currents to rat dorsal root ganglia modulates nerve injury-induced tactile allodynia.

BACKGROUND: Application of pulsed radiofrequency (PRF) currents to the dorsal root ganglia (DRG) has been reported to produce relief from certain pain states without causing thermal ablation. In this study, we examined the direct correlation between PRF application to DRG associated with spinal nerve injury and reversal of injury-induced behavioral hypersensitivity in a rat neuropathic pain model. METHODS: Neuropathic lesioning was performed via left L5 spinal nerve ligation on male adult Sprague-Dawley rats. Once the injured rats had developed tactile allodynia, one group was then assigned to PRF treatment of the L5 DRG and another group was assigned to the sham treatment to the DRG. Behavioral testing was performed on both the control and treated paws using the von Frey filament test before the surgery and at indicated days. The resulting data were analyzed using a linear mixed model to assess the overall difference between the treatment groups and the overall difference among the study days. Cohen's d statistic was computed from paired difference-from-baseline scores for each of the 14 study days after treatment and these measures of effect size were then used to descriptively compare the recovery patterns over time for each study group. RESULTS: Spinal nerve injury resulted in the development of behavioral hypersensitivity to von Frey filament stimulation (allodynia) in the hindpaw of the left (injury) side. Mixed linear modeling showed a significant difference between the treatment groups (P = 0.0079) and a significant change of paw withdrawal threshold means over time (P = 0.0006) for all 12 animals. Evaluation of Cohen's d (effect size) revealed that the PRF-treated animals exhibited better recovery and recorded larger effect sizes than the sham-treated animals on 10 of the 14 post-PRF treatment days and exhibited moderate-to-strong effects posttreatment at days 8 to 10 and at and beyond day 32. CONCLUSIONS: Findings from this study support that PRF of the DRG causes reversal of nerve injury (spinal nerve ligation)-induced tactile allodynia in rats. This allodynia reversal indicates that nonablative PRF acting via modulation of the DRG can speed recovery in nerve injury-induced pain.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second-order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI-ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R-), inhibitory (F-), and primary afferent (C-) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R-profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post-CCI-ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states.

Induction of Endoplasmic Reticulum Stress by Cadmium and Its Regulation on Nrf2 Signaling Pathway in Kidneys of Rats / 生物医学与环境科学（英文）

OBJECTIVE@#This study was conducted to investigate the regulation of endoplasmic reticulum stress on Nrf2 signaling pathway in the kidneys of rats.@*METHODS@#Rats were divided into twelve groups of six animals each. Some groups were pre-administered with bacitracin or tauroursodeoxycholic acid (TUDCA), and all of them were treated with 5-20 μmol/kg cadmium (Cd) for 48 h. The oxidative stress levels were analyzed using kits. The mRNA and protein expression levels of endoplasmic reticulum stress-related factors and Nrf2 signaling pathway-related factors were determined using RT-PCR and western blot.@*RESULTS@#Cd exposure resulted in oxidative stress in the kidneys of rats and upregulated the expression of endoplasmic reticulum stress (ERS)-related factors and Nrf2 signaling pathway-related factors, especially at doses of 10 and 20 μmol/kg Cd, and the expression changes were particularly obvious. Moreover, after pretreatment with bacitracin, Cd upregulated the expression of ERS-related factors to a certain extent and, at higher doses, increased the mRNA expression of Nrf2. After pretreatment with TUDCA, Cd reduced the level of ERS to a certain extent; however, at these doses, there were no significant changes in the expression of Nrf2.@*CONCLUSION@#Cadmium can result in ERS and oxidative stress in the kidneys of rats, activate Nrf2, and upregulate the transcriptional expression of phase II detoxification enzymes under these experimental conditions. ERS has a positive regulation effect on Nrf2 signaling pathway but has little effect on the negative regulation of Nrf2 signaling pathway in cadmium toxicity.

A novel analgesic isolated from a traditional Chinese medicine.

BACKGROUND: Current pain management is limited, in particular, with regard to chronic pain. In an attempt to discover novel analgesics, we combined the approach developed to characterize traditional Chinese medicine (TCM), as part of the "herbalome" project, with the reverse pharmacology approach aimed at discovering new endogenous transmitters and hormones. RESULTS: In a plant used for centuries for its analgesic properties, we identify a compound, dehydrocorybulbine (DHCB), that is effective at alleviating thermally induced acute pain. We synthesize DHCB and show that it displays moderate dopamine receptor antagonist activities. By using selective pharmacological compounds and dopamine receptor knockout (KO) mice, we show that DHCB antinociceptive effect is primarily due to its interaction with D2 receptors, at least at low doses. We further show that DHCB is effective against inflammatory pain and injury-induced neuropathic pain and furthermore causes no antinociceptive tolerance. CONCLUSIONS: Our study casts DHCB as a different type of analgesic compound and as a promising lead in pain management.

Exposure of the dorsal root ganglion to pulsed radiofrequency current in a neuropathic pain model of peripheral nerve injury.

The spinal nerve ligation model of neuropathic pain in rats, as originally described by Kim and Chung (Pain 50:355-363, 1992), provides an excellent venue to study the antinociception and modulation effects of pulsed radiofrequency (PRF) current in pain processing. We describe the procedure of application of PRF current near the exposed L5 dorsal root ganglion (DRG) in rats with L5 spinal nerve ligation injury-induced behavioral hypersensitivity. This method employs the direct visualization of the L5 DRG, allowing for confirmation of the location of the PRF probe adjacent to the DRG.

Simultaneous determination of cyclohexane and methylcyclohexane in workplace air by solvent desorption-gas chromatography / 中国职业医学

OBJECTIVE: To explore an improved method for determination of cyclohexane and methylcyclohexane in workplace air by solvent desorption-gas chromatography. METHODS: Cyclohexane and methylcyclohexane in workplace air were collected by activated carbon tubes,desorbed with carbon disulfide,separated by DB-1 capillary chromatography column,detected by flame ionization detector and quantified using the standard calibration curves. RESULTS: The linear range of the concentration of cyclohexane and methylcyclohexane were 1. 0-1 402. 2 and 0. 8-1 999. 4 mg / L respectively.Both the correlation coefficients were 0. 999 9. Both the detection limits were 0. 3 mg / L. The limits of quantification were1. 0 and 0. 8 mg / L respectively. Both the minimum detectable concentrations were 0. 2 mg / m3. The minimum quantitative mass concentrations of cyclohexane and methylcyclohexane were 0. 7 and 0. 6 mg / m3respectively( sample volume was 1. 5L). The average desorption efficiencies were 98. 5%-99. 3% and 97. 6%-99. 0% respectively. The relative standard deviations( RSD) of within-run precision were 0. 36%-0. 59% and 0. 34%-0. 50% respectively. The RSD of between-run precision were 0. 89%-2. 04% and 0. 87%-2. 22% respectively. The samples could be stored for up to 7 days at room temperature. CONCLUSION: This method has features of simple operation,high sensitivity and good precision,which is suitable for simultaneous determination of cyclohexane and methylcyclohexane in workplace air.

Determining indium in human whole blood,serum and urine by inductively coupled plasma-mass spectrometry / 中国职业医学

OBJECTIVE: To establish a methodology for determining indium in human whole blood,serum and urine by inductively coupled plasma-mass spectrometry( ICP-MS). METHODS: The whole blood,serum and urine samples were diluted 10 times in 0. 01%( mass fraction) Triton X-100 plus 0. 50%( mass fraction) nitric acid solution,and the indium level was determined by ICP-MS. Rhodium standard solution was used as the internal standard control. RESULTS: The working curve obtained from measurement of whole blood,serum and urine of normal individuals was compared to the standard curve and showed no significant difference in quantitative analysis( P > 0. 05). The linearity range of indium concentration in whole blood,serum and urine was 0. 000-20. 000 μg / L,and all the correlation coefficients were greater than 0. 999 with a detection limit of 0. 144 μg / L. The recovery rates of whole blood,serum and urine were 87. 90%-95. 92%,91. 50%-94. 20% and 90. 40%-96. 57%,respectively. The relative standard deviations( RSDs) of within-run precision were 3. 81%-7. 05%,3. 75%-5. 90% and 4. 31%-6. 62%,respectively. The RSDs of between-run precision were 2. 90%-7. 10%,3. 80%-5. 92% and 4. 16%-5. 94%,respectively. Samples could be stored for at least 14 days under the temperature of- 20 ℃. The indium in whole blood,serum and urine of workers occupationally exposed to indium( exposure group,135 person-time) and control group workers( 120 person-time) were examined. Indium was detected for 17 person-time in whole blood and serum in the exposure group with a detection rate of 1. 26%. Indium was not detected in urine samples in exposure group. It was not detected in all samples in control group. CONCLUSION: This methodology has features of simple operation,high accuracy and good precision,which is suitable for the accurate quantitative analysis of indium in biological samples.

Effect of DNA single strand breaks and 8-OHd G production on cadmium-induced toxicity on human embryonic kidney epithelial cells / 中国职业医学

OBJECTIVE: To explore the effects of cadmium chloride( CdCl_2) on DNA single strand breaks and the production of 8-hydroxy-2'-deoxyguanosine( 8-OHdG) in human embryonic kidney epithelial cells( HEK cells). METHODS: HEK cells in logarithm growth phase were divided into 5 groups and incubated with the different concentrations of CdCl_2( 0. 0,2. 5,5. 0,10. 0 and 20. 0 μmol/L) for 24,48 and 72 hours in vitro. After harvesting the cells,DNA single strand breaks was tested by single cell gel electrophoresis,and the level of 8-OHdG was measured using the enzyme linked immunosorbent assay. RESULTS: The Olive tail moment was statistically significant in the main effect of CdCl_2 exposed HEK cells( P < 0. 01). Among them,when HEK cells were exposed to 5. 0 μmol / L of CdCl_2,the Olive tail moment began to have a statistical significant increasing trend compared with the 0. 0 μmol / L group( P < 0. 05); when CdCl_2 concentration was 2. 5-10. 0 μmol / L,the Olive tail moment lengthened with the increasing dose of cadmium exposure,showing a doseeffect relationship( P < 0. 05). The tail DNA% was statistically significant in the interaction between exposure treatment and exposure time in HEK cells( P < 0. 01). Among them,when CdCl_2 concentration was at 2. 5-10. 0 μmol / L at 24 hours time point and 5. 0-20. 0 μmol / L at 48 hours time point,the tail DNA% raised with the increasing dose of cadmium exposure,showing a dose-effect relationship( P < 0. 05). The tail DNA% at 3 time points of 24,48 and 72 hours after exposure to 20. 0 μmol / L of CdCl_2 in HEK cells increased with the increasing time of cadmium exposure,showing a timeeffect relationship( P < 0. 05). The level of 8-OHdG had statistical significance in the main effect of CdCl_2 exposure treatment in HEK cells( P < 0. 05). Among them,the level of 8-OHdG was first significantly increased only after exposure to 10. 0 μmol / L CdCl_2 compared with the 0. 0 μmol / L group( P < 0. 05). After treatment with Ca Cl2,there was no doseeffect relationship and time-effect relationship found between the cadmium chloride exposure and tail length as well as the tail / head length ratio and 8-OHdG level. CONCLUSION: To a certain extent,CdCl_2 exposure may cause both DNA single strand breaks and 8-OHdG production in HEK cells. Compared with 8-OHdG,the DNA single strand breaks show more significant change with a lower dose of cadmium treatment,which may be related to its higher sensitivity to cadmium toxicity than 8-OHdG.

Profiling of dynamically changed gene expression in dorsal root ganglia post peripheral nerve injury and a critical role of injury-induced glial fibrillary acidic protein in maintenance of pain behaviors [corrected].

To explore cellular changes in sensory neurons after nerve injury and to identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may also play a critical, but yet identified, role in the maintenance of neuropathic pain states.

Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation.

Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage-gated calcium channel (VGCC) alpha2delta1 subunit (Ca(v)alpha2delta1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Ca(v)alpha2delta1 in neuronal tissues. Ca(v)alpha2delta1 overexpression resulted in enhanced currents, altered kinetics and voltage-dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue-injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Ca(v)alpha2delta1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration-dependently in transgenic, but not wild-type, sensory neurons. Thus, elevated neuronal Ca(v)alpha2delta1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.