Objective: Electroacupuncture (EA) is an alternative treatment option for pain. Different frequencies of EA have different pain-relieving effects; however, the central mechanism is still not well ...understood. Methods: The FosTRAP:Ai9 mice were divided into three groups (sham, 2 Hz, and 100 Hz). The mice were intraperitoneally injected with 4-hydroxytamoxifen (4-OHT) immediately after EA at Zusanli (ST36) for 30 min to record the activated neurons. One week later, the mice were sacrificed, and the number of TRAP-treated neurons activated by EA in the thalamus, amygdala, cortex, and hypothalamus was determined. Results: In the cortex, 2 Hz EA activated more TRAP-treated neurons than 100 Hz EA did in the cingulate cortex area 1 (Cg1) and primary somatosensory cortex (S1), and 2 Hz and 100 Hz EAs did not differ from sham EA. TRAP-treated neurons activated by 2 Hz EA were upregulated in the insular cortex (IC) and secondary somatosensory cortex (S2) compared with those activated by 100 Hz and sham EA. In the thalamus, the number of TRAP-treated neurons activated by 2 Hz EA was elevated in the paraventricular thalamic nucleus (PV) compared with those activated by sham EA. In the ventrolateral thalamic nucleus (VL), the number of TRAP-treated neurons activated by 2 Hz EA was significantly upregulated compared with those activated by 100 Hz EA, and sham EA showed no difference compared to 2 Hz EA or 100 Hz EA. TRAP-treated neurons were more frequently activated in the ventral posterolateral thalamic nucleus (VPL) by 2 Hz EA than by 100 Hz or sham EA. Conclusion: Low-frequency EA ST36 effectively activates neurons in the Cg1, S1, S2, IC, VPL, PV, and VL. The enhanced excitability of the aforementioned nuclei induced by low-frequency EA may be related to its superior efficacy in the treatment of neuropathological pain.
Background: Acute pain can transition to chronic pain, presenting a major clinical challenge. Electroacupuncture (EA) can partly prevent the transition from acute to chronic pain. However, little is ...known about the mechanisms underlying the effect of EA. This study investigated the effect of EA on pain transition and the activation of metabotropic glutamate receptor 5 (mGluR5)-protein kinase C epsilon (PKCepsilon) signaling pathway in the dorsal root ganglia (DRG). Methods: The hyperalgesic priming model was established by the sequential intraplantar injection of carrageenan (1%, 100 microL) and prostaglandin E2 (PGE2) into the left hind paw of rats. EA treatment (2/100 Hz, 30 min, once/day) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints in rats. Von Frey filaments were used to investigate the mechanical withdrawal threshold (MWT) at different time points. The protein expression levels of mGluR5 and PKCepsilon in the ipsilateral L4-L6 DRGs of rats were detected by Western blot. Some pharmacological experiments were performed to evaluate the relationship between mGluR5, PKCepsilon and the MWT. It was also used to test the effects of EA on the expression levels of mGluR5 and PKCepsilon and changes in the MWT. Results: Sequential injection of carrageenan and PGE2 significantly decreased the MWT of rats and up-regulated the expression level of mGluR5 and PKCepsilon in the ipsilateral L4-L6 DRGs. EA can reverse the hyperalgesic priming induced by sequential injection of carrageenan/PGE and down-regulate the protein expression of mGluR5 and PKCepsilon. Glutamate injection instead of PGE2 can mimic the hyperalgesic priming model. Pharmacological blocking of mGluR5 with specific antagonist MTEP can prevent the hyperalgesic priming and inhibit the activation of PKCepsilon in DRGs. Furthermore, EA also produced analgesic effect on the hyperalgesic priming rats induced by carrageenan/mGluR5 injection and inhibited the high expression of PKCepsilon. Sham EA produced none analgesic and regulatory effect. Conclusion: EA can regulate pain transition and it may relate with its inhibitory effect on the activation of mGluR5-PKCepsilon signaling pathway in the DRGs. Keywords: electroacupuncture, pain transition, hyperalgesic priming, mGluR5, PKCepsilon, DRGs
Acute pain can transition to chronic pain, presenting a major clinical challenge. Electroacupuncture (EA) can partly prevent the transition from acute to chronic pain. However, little is known about ...the mechanisms underlying the effect of EA. This study investigated the effect of EA on pain transition and the activation of metabotropic glutamate receptor 5 (mGluR5)-protein kinase C epsilon (PKCε) signaling pathway in the dorsal root ganglia (DRG).
The hyperalgesic priming model was established by the sequential intraplantar injection of carrageenan (1%, 100 μL) and prostaglandin E2 (PGE2) into the left hind paw of rats. EA treatment (2/100 Hz, 30 min, once/day) was applied at bilateral Zusanli (ST36) and Kunlun (BL60) acupoints in rats. Von Frey filaments were used to investigate the mechanical withdrawal threshold (MWT) at different time points. The protein expression levels of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs of rats were detected by Western blot. Some pharmacological experiments were performed to evaluate the relationship between mGluR5, PKCε and the MWT. It was also used to test the effects of EA on the expression levels of mGluR5 and PKCε and changes in the MWT.
Sequential injection of carrageenan and PGE2 significantly decreased the MWT of rats and up-regulated the expression level of mGluR5 and PKCε in the ipsilateral L4-L6 DRGs. EA can reverse the hyperalgesic priming induced by sequential injection of carrageenan/PGE and down-regulate the protein expression of mGluR5 and PKCε. Glutamate injection instead of PGE2 can mimic the hyperalgesic priming model. Pharmacological blocking of mGluR5 with specific antagonist MTEP can prevent the hyperalgesic priming and inhibit the activation of PKCε in DRGs. Furthermore, EA also produced analgesic effect on the hyperalgesic priming rats induced by carrageenan/mGluR5 injection and inhibited the high expression of PKCε. Sham EA produced none analgesic and regulatory effect.
EA can regulate pain transition and it may relate with its inhibitory effect on the activation of mGluR5-PKCε signaling pathway in the DRGs.
Pain memory is commonly considered an underlying cause of chronic pain and is also responsible for a range of anxiety. Electroacupuncture (EA) has been shown to ameliorate pain memories and exert ...anti-anxiety effects. Previous research has indicated that GABAergic neurons and/or GABA receptors (GABARs) in the midcingulate cortex (MCC) have potential associations with chronic pain and anxiety. However, there is no known empirical research that has specifically studied the effects of EA on the GABAergic system in the MCC. Here, we used cross-injection of carrageenan to establish the pain memory rats model. Immunofluorescence were used to detect the excitability of GABAergic neurons within MCC. Von Frey filament, elevated zero maze, and open field tests were used to measure mechanical allodynia and anxiety-like behaviors, combined with chemogenetic and pharmacologic technologies. Finally, this study provides evidence that pain memories contribute to generalized negative emotions and that downregulating the activity of GABAergic neurons within MCC could block pain memories and reverse anxiety emotion. Specifically, GABA
R is involved in pain memory and related anxiety-like behaviors. Activation of GABAergic neurons in the MCC did not reverse the effects of EA on pain memories and related anxiety-like behaviors, whereas these effects could be reversed by a GABA
R agonist. These findings highlight the functional significance of GABA
R in the EA-mediated attenuation of pain memories and related anxiety-like behaviors in rats.
A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the ...first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185-900 mOsmol kg-1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 +/- 0.39 microns 3 (mean +/- SEM; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r2 = 0.99) with an osmotically inactive cell fraction of 67.4 +/- 4.5%. The rate of water permeability (Lp) was determined to be 1.03 +/- 0.05 microns min-1 atm-1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30 degrees C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8 degrees C and 0 degree C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg-1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg-1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg-1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg-1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.
Pain is considered a multidimensional conscious experience that includes a sensory component and a negative affective-motivational component. The negative affective-motivational component of pain is ...different from the sensory component and amplifies the pain experience. Nowadays, a significant number of preclinical research groups have focused their attention on the affective symptoms of pain. In the present study, we investigated the pain aversion and anxiety-like behavior of the complete Freund's adjuvant (CFA)-induced chronic pain model. CFA rats experienced spontaneous pain during pain-paired conditioning (pain aversion) and spontaneous pain produces an affective response (anxiety-like behavior). Moreover, pain aversion was gradually attenuated, while the anxiety-like behavior increased in 4 weeks. Therefore, although the negative effect (including pain aversion and anxiety) is always associated with hyperalgesia, the manifestations of negative effect may follow different time courses, which may influence the progress of primary disease. The findings illustrate that targeted therapy should focus on a specific aspect in different stages of pain. Our study emphasizes the necessity of using multiple tests to study pain comorbidities.
The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at ...which spermatozoa are collected, (ii) the cooling rate to 0 degrees C and (iii) the warming rate from -196 degrees C to ambient. When excised epididymides were cooled to near 0 degrees (1-4 degrees C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8-10 times higher than when the spermatozoa were collected from the epididymides at 22 degrees C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000 degrees C min-1 were about five times higher than those in suspensions warmed at about 7500 degrees C min-1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6-8% when the collection temperature was 22 degrees C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from -196 degrees C. When spermatozoa collected at 22 degrees C were abruptly cooled to 0 degrees C, 40-80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0 degrees C at 1 degree C min-1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75-90%).
Experiments were conducted to determine the water volume and osmotic behaviour of mouse spermatozoa using an electron paramagnetic resonance technique using the spin label tempone, and the broadening ...agent potassium chromium oxalate. After a swim-up procedure, an average water volume of 43.3 micron3 of individual spermatozoa was obtained at 290 mosmol. If a water compartment of 59% is assumed, the total volume of mouse spermatozoa is 73.4 micron3. A plot of the relative water volume of mouse spermatozoa versus the reciprocal of buffer osmolality (Boyle van't Hoff plot) is linear in the range 250-900 mosmol of sodium chloride solutions (r2 = -.96). The Boyle van't Hoff plot intercept indicates that 13% of the spin-label accessible isotonic water is osmotically inactive.
The permeability of human spermatozoa to glycerol and its activation energy were determined using electron paramagnetic resonance (EPR) techniques. EPR was used to monitor the aqueous cell volume ...change vs. time during the glycerol permeation process using the aqueous spin label 15N-tempone and the membrane impermeable broadening agent potassium trioxalatochromiate (chromium oxalate). The permeation process was completed in tens of seconds, requiring the use of a stopped-flow methodology. The glycerol permeability coefficient (Pg) was determined by fitting a simple theoretical model to the experimental data. The permeabilities of human spermatozoa in 1 molar and 2 molar glycerol at 20 degrees C are (10.3 +/- 0.3).10(-4) cm/min (mean +/- S.D.) and (6.0 +/- 1.4).10(-4) cm/min, respectively. The permeabilities of human spermatozoa in 2 molar glycerol at 30, 20, 10, and 0 degrees C are (8.3 +/- 1.3).10(-4) cm/min, (6.0 +/- 1.4).10(-4) cm/min, (2.1 +/- 0.4).10(-4) cm/min, and (1.1 +/- 0.3).10(-4) cm/min, respectively. The activation energy (Ea) for glycerol permeation between 30 degrees C and 0 degrees C was found to be 11.6 kcal/mol.
An electron spin resonance technique using the spin label tempone and the broadening agent potassium chromium oxalate was used to measure the water volume of human sperm. The toxicity of tempone (5 ...mmol/L) and potassium chromium oxalate (50 mmol/L) to sperm was measured over a time span of 120 minutes using computer‐assisted semen analysis. Tempone had no effect on any computer‐assisted semen analysis parameters, including motility. Potassium chromium oxalate reduced sperm motility by an average of 24% during the first 30 minutes of exposure. After selection by swim‐up and correction for the presence of dead cells and cytoplasmic droplets, a water volume of 20.0 ± 2.9 μ3 was obtained. This yields a total volume of 33.9 μ3 if a water compartment of 59% by volume is assumed. These results are consistent with other shape‐independent techniques for measuring volume, but larger than the generally accepted optical and electronic particle counter sizes.