Although labor pain is treated clinically by the administration of local anesthetics alone or with opioids in the epidural or spinal spaces (1-4), however, the mechanisms of labor pain mechanisms have not been fully elucidated during to the lack of animal models, and the side effects of drugs still existed. For these reasons, new, more straight forward approaches to the treatment of labor pain are essential. Advances in understanding and treatment for labor pain care, in general, hampered by our limited understanding of visceral pain because most laboratory work has focused on somatic pain. Recently, an acute uterine cervical distension (UCD) model in the lightly anesthetized rat has been used in several reports to study the physiology of acute visceral nociception induced by the distending of the uterine cervix (5,6). UCD produces a stimulus-dependent increase in afferent nerve and reﬂex abdominal muscle construction. Besides, local infiltration of the cervix with lidocaine can inhibit the expression of Fos in the spinal cord induced by UCD (7). For protein, the product of c-fos immediate early gene (IEG), has been used as a marker for neuronal activation in center nerve system, further suggesting that UCD is a noxious stimulus and reflects noxious input from the uterine cervix rather than traction on other visceral or somatic structures by deformation of the tissue during parturition. Therefore, this acute UCD model can be used as a visceral model related to labor pain.
Spinal microglia can be activated or mobilized under several pain states (8-11) and plays a vital role in the transmission of nociceptive signals by releasing many inflammatory mediators (IFMs), such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and chemokines. Saab et al. found activated microglia in a rat model with chronic visceral hyperalgesia induced by neonatal colon irritation by repeated colorectal distension using an angioplasty balloon. Using minocycline to interrupt microglia activation and its associated release of proinﬂammatory mediators would reverse the visceral pain behavior and restore the standard processing of neuronal information along the visceral pain pathway in adulthood. The content of inflammatory cytokines in cerebrospinal fluid increased significantly during parturition, suggested microglia may be involved in the labor pain signals integration as well. However, the exact role of spinal microglia in acute uterine cervical distension is the primary unknown.
The current labor analgesia method can be divided into non-pharmacological analgesia and pharmacological analgesia. The side effects of ordinarily used drugs, local anesthetics, and opioids, include itching, respiratory inhibition of maternal and infant, hypotension, disturbance of parturient, and motor blockade. Non-drug analgesia such as acupuncture point stimulation, ramaze therapy, has smaller trauma, no drug side effect, less disturbance of parturient. But the analgesic effect of acupuncture point stimulation is not ideal, but existing pieces of evidence show acupuncture point stimulation can reduce perioperative opioid consumption. So, the analgesia methods may improve the labor analgesia effect and reduce the side effect by reducing the consumption of anesthetics.
In our study, we investigated the effects of acupuncture point stimulation in labor analgesia and the activation of spinal microglia in acute uterine cervical distension rats.
We present the following article in accordance with the ARRIVE reporting checklist (available at http://dx.doi.org/10.21037/apm-20-693).
Sprague-Dawley adult virgin female rats, weighting 220–280 g at the time of testing, were purchased from the Laboratory Animal Center, Academy of Medical Sciences of Zhejiang Province. All animals were kept in separate cages at 12/12 h with circadian rhythm inverted, and the temperature was 24–28 °C, the relative humidity was 50%±5%. Food, but not water, was withheld for 12 h before the study. The experimental animal ethics committee approved all procedures of Wenzhou Medical University and consistent with the ethical guidelines for the investigation of experimental pain in animals.
The study was divided into three parts. UCD was applied topically with a distension force of 75 g. In the ﬁrst part, to investigate the activation of microglia in the spinal cord, 24 rats were randomly divided into the standard group, sham group, and UCD group. The effect of time of UCD was studied by UCD distension of 30 min (UCD 30, n=8), 60 min (UCD 60, n=8) or 90 min (UCD 90, n=8). The T12 to L2 spinal cord segments were removed for immunohistochemical analysis (n=4 at each time point).
The second part of the study aims to detect whether microglia activation contributes to acute uterine cervical distension. After implantation of the intrathecal catheter, 16 rats were randomly divided into two groups. The rats received an intrathecal injection of PBS or minocycline before UCD (n=8 per group). The T12 to L2 spinal cord segments were removed for immunohistochemical analysis (n=4 at each time point).
In the third part of the study, to study the role of acupuncture affect the activation of microglia in the spinal cord by acute uterine cervical distension in rats. Sixteen rats were randomly divided into the acupuncture group and non-acupuncture group (n=8 per group). The T12 to L2 spinal cord segments were removed for immunohistochemical analysis (n=4 at each time point).
Uterine cervical distension
Animals were anesthetized with intraperitoneal chloral hydrate 300 mg/kg and spontaneous ventilation. A small low abdominal laparotomy was performed to expose the uterus, and two ﬁne metal rods were inserted through both uterine cervical losses. The rods entered from the uterus and left through the vaginal wall, both as near as possible to the cervix. With one rod attached to a metal stand by a silk suture, UCD was applied to the cervix by hanging various standard weights on another rod through a silk suture and pulley. The sham operation group included rats with only an abdominal incision and two metal rods inserted through cervical losses but without distension (0 g). All skin incisions were anesthetized with 1% lidocaine for local infiltration. Body temperature was supported in the range of 37–38 °C throughout the experiment with a circulating-water heating pad (11).
The visceromotor response induced by UCD was recorded by quantifying electromyographic (EMG) activity with a concentric needle electrode inserted into the right rectus abdominis superior to the inguinal ligament. Through the Medlab-4C501 (Anhui Zhenghua biological instrument equipment Co. Ltd., Anhui, China) biological signal collection system, EMG activity was continuously ampliﬁed (10,000 gain), ﬁltered (high pass, 500–5,000 Hz), digitized and stored for off-line analysis. Rats were subjected to UCD stimulus (25, 50, 75, and 100 g, respectively, for 10 s) or no distension (sham group, 0 g) (n=8 for each distension force) following a 5-min basal EMG recording. Each UCD force was only applied once for each individual rat. The raw EMG data over the 10 s just before (baseline) and during UCD was rectiﬁed and quantiﬁed by calculating the area under the curve. For data analysis, the percentage of are under the curve of distension to baseline was used as there sponses to UCD for each animal. The relevant data in the sham group also were calculated.
Intrathecal drug administration
Intrathecal catheters were inserted according to the method of Yaksh and Rudy (5) with a slight modiﬁcation. In brief, a polyethylene PE10 catheter was implanted in the lumbar subarachnoid space of each rat during deep pentobarbital sodium (40 mg/kg, i.p.) anesthesia. Animals were allowed 5–7 days recovery and were euthanized at once if they showed any signs of motor deﬁcit.
Data were expressed as mean ± standard error of means (SEM). In the ﬁrst part and the second part, two sets of data were compared with the t-test (paired or unpaired when proper). In the third part, the statistical analysis was performed using one-way ANOVA with the use of a factorial design with SPSS19.0, and the statistical figures were all performed using Graph Pad Prism 5.0 software. For all analyses, the values of P<0.05 were statistically signiﬁcant.
EMG response to UCD, and IBA1 expression (Figures 1,2).
Sixty min after UCD, compared with the sham group and the standard group, the EMG of the UCD group was higher (P<0.05), but no difference was observed between standard group and sham group (P>0.05).
The effects of minocycline and PBS on cervical, uterine distension to study EMG and IBA1 expression (Figures 3,4).
The effects of acupuncture and non-acupuncture on cervical, uterine distension to study EMG and IBA1 expression (Figures 5,6).
Our study found that UCD accompanied by activation of spinal microglia, and the duration of UCD exert the number of activated microglia signiﬁcantly. Intrathecal injection of minocycline and acupuncture point stimulation depressed the UCD induced enhancement of EMG and reduced the activation of spinal microglia.
The role of microglia in somatic pain has been extensively studied, while the data about the visceral pain are quite limited. Our study found that UCD activated microglia. Our results are consistent with the result of earlier studies showing the inflammation, colorectal distension, and chronic psychological stress, which induced chronic visceral pain, can activate the spinal microglia as well. Notably,UCD induced acute visceral pain but not chronic visceral pain. Although the pain processes between chronic and acute nociception have much in common, they also have several significant differences. UCD produces a force-dependent increase in neural activity in the hypogastric nerve, evoke abdominal muscle reflex electromyographic activity (12) reminiscent of the sacral neural activity and reﬂex electromyographic activity evoked by noxious colorectal distension (13). Systemic opioids inhibit UCD-induced reﬂex electromyography and afferent ﬁber ﬁring in doses thought to be antinociceptive in rats. Our study also found that the stimulus intensity of UCD was strictly related to the activation of microglia, and intrathecal injection of minocycline effectively blocked UCD induced activation of microglia and acute visceral pain. Thus, these results suggest activation of spinal microglia also contributes to the initiating of acute visceral pain.
Our study suggests an increase in spinal microglia expression with increasing duration of UCD, although we cannot exclude the inﬂuence of more significant time from onset of UCD to perfusion in the UCD 60-min group compared with the UCD 30-min group to increase microglia expression.
The mechanism of microglia contribute to the acute visceral pain is still unknown. UCD is a model for analogous labor pain; the previous study has shown that the content of inflammatory cytokines in cerebrospinal fluid increased significantly during parturition in human beings. It is well known that the role of microglia in pain signals integration mainly rely upon releasing many IFMs, such as IL-1, TNF-α, PGE2, and chemokines, and the activated microglia is considered a primary source of the inflammatory cytokines. Under chronic pain conditions, P-p38 MAPK and ERK5 are activated in spinal microglia, and the hyperalgesia can be reversed by minocycline, p38 inhibitor, or knockdown of ERK5. It has been shown that p-p38 MAPK and ERK5 pathway can be activated in an hour by the nociceptive stimulus and plays a pivotal role in regulating the expression of IFMs in spinal (14). In the current study, activation of spinal microglia peaked at 60 min, with a slow decline but remained increased at 120 min after UCD, suggests that UCD induced activation of microglia may release IFMs, and modulate the visceral pain signals in the spinal cord.
The mechanism of acupuncture regulates visceral pain: spinal cord is the first level of pain signals after entering the central integration center, is the nociceptive information transmission from relay station, which can direct modulation of pain, and receives signals central downward adjustment. The spinal cord is a vital part of the pain stimulus to adjust to the visceral pain. It is believed that the spinal cord dorsal horn accepted by many pain downward modulation systems composed of pulp in the central regulation of visceral injury information. By multiple neural spinal cords, such as PAG, amygdala, descending pain modulation system composed of cingulate, the dorsal horn of spinal cord conduction of pain information has a strong inhibitory effect, and acupuncture may be activated descending pain modulation system and exert its analgesic effect. Recent research shows that through the spinal cord dorsal horn neurons, message to the pulp in the central, acupuncture letter to activate pain downward modulation system, and then to the dorsal horn of c-Fos, P38, NRI and 5-HT receptor expression, which in turn have an analgesic effect. Further study shows that the body noxious stimulation and acupuncture point acupuncture inhibitory effect on visceral nociceptive information transmission (15,16).
Participate in electric acupuncture treatment of visceral pain-related neurotransmitters are opioid, 5-HT, and other neurotransmitters and conditioning. A lot of research has shown that acupuncture to relieve visceral pain effect, most likely by activating endogenous opioid system and play a role, this is because the application of naloxone after acupuncture effect on visceral pain was canceled.
Acupuncture has the spinal good curative effect in the treatment of visceral sensitivity, and this may be through the spinal cord, the thalamus, the brain stem, and/or the cortex. The interaction between the senior center may also play a role by nerve endocrine immunity network.
In summary, this study supplies novel evidence that UCD stimulates spinal cord microglia expression, and acupuncture affects the activation of microglia. So, acupuncture can relieve labor pain, and that acupuncture might be useful in the treatment of such pain.
Funding: This work was supported by the Natural Science Foundation of Zhejiang Province (LY17C090002), Hangzhou Science and technology development plan project (20150733Q12) and Zhejiang medical and health science and technology program (2018KY138). We are very grateful to Wenzhou Medical University, part of our experiment was done under their help.
Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at http://dx.doi.org/10.21037/apm-20-693
Dada Sharing Statement: Available at http://dx.doi.org/10.21037/apm-20-693
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/apm-20-693). The authors have no conflicts of interest to declare.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The experimental animal ethics committee approved all procedures of the Wenzhou Medical University and consistent with the ethical guidelines for the investigation of experimental pain in animals (No. wydw2019-0927).
Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.
- Eisenach JC. Pain physiology and pharmacology: Clinical relevance (refresher course lecture). American Society of Anesthesiologists 2001;511:1-7.
- Zakowski M. Complications associated with regional anesthesia in the obstetric patient. Semin Perinatol 2002;26:154-68. [Crossref] [PubMed]
- Nelson KE, Rauch T, Terebuh V, et al. A comparison of intrathecal fentanyl and sufentanil for labor analgesia. Anesthesiology 2002;96:1070-3. [Crossref] [PubMed]
- Bucklin BA, Chestnut DH, Hawkins JL. Intrathecal opioids versus epidural local anesthetics for labor analgesia: a meta-analysis. Reg Anesth Pain Med 2002;27:23-30. [PubMed]
- Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031-6. [Crossref] [PubMed]
- Sandner-Kiesling A, Eisenach JC. Estrogen reduces efficacy of mu- but Not kappa-opioid agonist inhibition in response to uterine cervical distension. Anesthesiology 2002;96:375-9. [Crossref] [PubMed]
- Tong C, Ma W, Shin SW, et al. Uterine cervical distension induces cFos expression in deep dorsal horn neurons of the rat spinal cord. Anesthesiology 2003;99:205-11. [Crossref] [PubMed]
- Sandner-Kiesling A, Eisenach JC. Pharmacology of opioid inhibition to noxious uterine cervical distension. Anesthesiology 2002;97:966-71. [Crossref] [PubMed]
- Shin SW, Eisenach JC. Intrathecal morphine reduces the visceromotor response to acute uterine cervical distension in an estrogen-independent manner. Anesthesiology 2003;98:1467-71; discussion 6A.
- Shin SW, Sandner-Kiesling A, Eisenach JC. Systemic, but not intrathecal ketorolac is antinociceptive to uterine cervical distension in rats. Pain 2003;105:109-14. [Crossref] [PubMed]
- Gosselin RD, Suter MR, Ji RR, et al. Glial cells and chronic pain. Neuroscientist 2010;16:519-31. [Crossref] [PubMed]
- Saab CY, Wang J, Gu C, et al. Microglia: a newly discovered role in visceral hypersensitivity? Neuron Glia Biol 2006;2:271-7. [Crossref] [PubMed]
- Wang LZ, Liu X, Wu WX, et al. Phosphorylation of spinal signaling-regulated kinases by acute uterine cervical distension in rats. Int J Obstet Anesth 2010;19:50-5. [Crossref] [PubMed]
- Ness TJ, Gebhart GF. Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat. Brain Res 1988;450:153-69. [Crossref] [PubMed]
- Ji RR, Befort K, Brenner GJ, et al. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J Neurosci 2002;22:478-85. [Crossref] [PubMed]
- Pezet S, Malcangio M, Lever IJ, et al. Noxious stimulation induces Trk receptor and downstream ERK phosphorylation in spinal dorsal horn. Mol Cell Neurosci 2002;21:684-95. [Crossref] [PubMed]