Placental Barrier on Cadmium Transfer from Mother to Fetus in Related to Pregnancy Complications.

Purpose: As two of the most severe and common medical disorders during pregnancy, gestational diabetes mellitus (GDM) and hypertensive disorder complicating pregnancy (HDCP) cause adverse effects on placental barrier function and thus may lead to a high risk of intrauterine exposure to toxic metals from mother to fetus. This study investigates the impact of the placental barrier on the transfer of cadmium (Cd) from mother to fetus and the relationship between pregnancy complications. Methods: A total of 107 pairs of samples were collected in Kunming, China; 29 were from healthy pregnant women, and 78 were from patients with pregnancy complications. Cd was measured in each mother's placenta and maternal and umbilical cord blood. The expressions of MT and Cd-MT complex in blood and placental tissue samples were determined by enzyme-linked immunosorbent assay (ELISA). Results: The cesarean section rate in the whole pathological group (60.7%) was higher than that in the normal group (20.7%), and the ratio of the effective barrier (ratio of maternal blood to umbilical cord blood>1) in the pathological group (74%) was lower than that in the normal group (79%). In addition, the proportion of practical placental barriers in women aged 20-25 years was 83.3%, 76.3% in women aged 26-30 years, 74.3% in women aged 31-35 years, 70% in women aged 36-40 years, and 71% in women aged 40-45 years. The Cd content in the placenta of the three pathological groups was significantly higher than that in maternal and umbilical cord blood (P<0.05), and the distribution of Cd was the same as that in the normal group. However, there was no significant difference between maternal and umbilical cord blood Cd concentrations in the pathological group. The Cd concentration in the normal group's maternal blood was significantly higher than that in cord blood (P<0.05). In addition, the expression levels of both metallothionein (MT) and Cd-MT complex in placenta is much higher than in maternal and umbilical blood, and which in normal group are significantly higher than those in pathological group. Conclusion: Both mothers and fetuses are at increased health risk for pregnancy disorders when maternal age, BMI, or body weight increases. Increased maternal age increases the likelihood of Cd transfer from the mother to the fetus. Pregnancy complications may induce lower expression of MT, thus reducing the Cd-MT complex in the placenta, weakening the placental barrier, and increasing the risk of Cd transfer and exposure to the fetus.

Genetic Polymorphisms in microRNA Genes Targeting PI3K/Akt Signal Pathway Modulate Cervical Cancer Susceptibility in a Chinese Population.

Polymorphisms in microRNA (miRNA) genes could influence the expression of miRNAs that regulate the PI3K/Akt signalling pathway and play crucial roles in cancer susceptibility. To investigate the association of single nucleotide polymorphisms (SNPs) in miRNA genes of PI3K/Akt with cervical intraepithelial neoplasia (CIN) and cervical cancer (CC), nine SNPs located in miRNA genes were selected for genotyping, and the association of these SNPs with CIN and CC risk was evaluated. A total of 1,402 participants were enrolled in the current study, including 698 healthy individuals in the control group, 431 patients with CC, and 273 patients with CIN. Nine SNPs in miRNA genes (rs107822 in miR-219a, rs10877887 in let-7i, rs2292832 in miR-149, rs353293 in miR-143, rs3746444 in miR-499, rs3803808 in miR-132, rs4078756 in miR-10b, rs629367 in let-7a, and rs7372209 in miR-26a) were genotyped using MassArray, and the association of these SNPs with CIN and CC were analysed. The results showed that the frequencies of rs107822 in miR-219a and rs2292832 in miR-149 were significantly different between the control and CC groups (p < 0.005). The C allele of rs107822 in miR-219a was associated with an increased risk of CC (OR = 1.29, 95%CI:1.09-1.54) whereas the C allele of rs2292832 in miR-149 was associated with a decreased risk of CC (OR = 0.77, 95%CI:0.64-0.92). The results of inheritance model analysis showed that the best-fit inheritance models for rs107822 and rs2292832 were log-additive. The 2CC + CT genotype of rs107822 could be a risk factor for CC when compared with the TT genotype (OR = 1.28, 95%CI:1.08-1.51). The 2CC + CT genotype of rs2292832 could be a protective factor against CC when compared with the TT genotype (OR = 0.76, 95%CI:0.64-0.92). However, no association of these SNPs with CIN was found in the current study. Our results suggest that rs107822 in the promoter region of miR-219a and rs2292832 in pre-miR-149 region are associated with the risk of CC.

Association of Long Non-Coding RNAs (lncRNAs) ANRIL and MALAT1 Polymorphism with Cervical Cancer.

Background: Long non-coding RNAs (lncRNAs) and their polymorphisms play crucial roles in the development of different cancers. Methods: Eight single-nucleotide polymorphisms (SNPs) in ANRIL and MALAT1 (rs1333045, rs4977574, rs1333048, and rs10757278 in ANRIL and rs11227209, rs619586, rs664589, and rs3200401 in MALAT1) were enrolled and genotyped in a total of 1248 samples, including 587 patients with cervical cancer (CC) and 661 healthy individuals using in TaqMan assay. The association of these SNPs with CC was then evaluated. Results: Our results showed that the allele and genotype frequencies of rs3200401 in MALAT1 were significantly different between the control and CC groups after Bonferroni correction (P = 0.001 and P = 0.004, respectively), indicating that the C allele is a protective factor against CC (OR = 0.70; 95% CI = 0.57-0.87). In addition, the allele and genotype frequencies of rs4977574 in ANRIL were significantly different between the control and CC groups after Bonferroni correction (P = 0.004 and P = 0.014, respectively), and the A allele might be a protective factor for CC (OR = 0.80; 95% CI = 0.68-0.93). For subgroup analysis, the alleles of rs3200401 in MALAT1 showed significant differences between the control and adenocarcinoma (AC) and control and squamous cell carcinoma (SCC) groups (P = 0.005 and P = 0.004, respectively). The rs3200401C allele could be a protective factor for AC and SCC development (OR = 0.57; 95% CI = 0.38-0.85; OR = 0.72; 95% CI = 0.58-0.90). Moreover, the rs3200401C allele could be a protective factor for cervical cancer stage I development (OR = 0.67; 95% CI = 0.53-0.86). Conclusion: Our results indicate that rs3200401 in MALAT1 and rs4977574 in ANRIL could play key roles in the CC development.

Pregnancy complications effect on the nickel content in maternal blood, placenta blood and umbilical cord blood during pregnancy.

BACKGROUND: Nickel (Ni) may accumulate in the human body and has biological toxicity and carcinogenicity. Ni has an extensive impact on the health of pregnant women and fetuses during gestation. AIM: To evaluate Ni exposure in pregnant women in Kunming, Yunnan Province, China; to describe the distribution of Ni in the maternal-fetal system and placental barrier function; and to investigate the effect of Ni exposure on fetal health in mothers with pregnancy complications. METHODS: Seventy-two pregnant women were selected using a case-control design. The women were divided into two groups: The control group (no disease; n = 29) and the disease group [gestational diabetes (GDM), hypertensive disorder complicating pregnancy (HDCP), or both; n = 43]. The pregnant women in the disease group were further divided as follows: 14 cases with GDM (GDM group), 13 cases with HDCP (HDCP group) and 16 cases with both GDM and HDCP (disease combination group). Basic information on the pregnant women was collected by questionnaire survey. Maternal blood, placenta blood and cord blood were collected immediately after delivery. The Ni content in paired samples was determined using inductively coupled plasma mass spectrometry. RESULTS: Compared to the control group, age was higher and body mass index was greater in pregnant women in the disease groups (28.14 ± 2.54 vs 28.42 ± 13.89, P < 0.05; 25.90 ± 3.86 vs 31.49 ± 5.30, P < 0.05). The birth weights of newborns in the HDCP group and the control group were significantly different (2.52 ± 0.74 vs 3.18 ± 0.41, P < 0.05). The content of Ni in umbilical cord blood in the entire disease group was higher than that in the control group (0.10 ± 0.16 vs 0.05 ± 0.07, P < 0.05). CONCLUSION: In the maternal-fetal system of women with pregnancy complications, the barrier effect of the placenta against Ni is weakened, thus affecting healthy growth of the fetus in the uterus.

miR­199a­3p suppresses cervical epithelial cell inflammation by inhibiting the HMGB1/TLR4/NF­κB pathway in preterm birth.

Preterm birth (PTB) is the primary cause of neonatal mortality worldwide. Infection and inflammation are considered to be the primary causes of PTB. Cervical remodeling is an important step in the process of preterm delivery, and the destruction of the cervical epithelial barrier and inflammation are important triggers of cervical remodeling. The aim of the present study was to determine the effect and underlying mechanism of microRNA (miR)­199a­3p/high­mobility group box 1 protein (HMGB1) signaling in cervical epithelial inflammation in PTB. The results of this study revealed that miR­199a­3p was significantly decreased in cervical epithelial tissue samples from patients in both the preterm labor and preterm premature rupture of membrane groups. This decrease was also observed in tissue samples from a lipopolysaccharide (LPS)­induced PTB mouse model and in LPS­induced ectocervical and endocervical cells. Whereas, the expression of HMGB1 and toll­like receptor 4 (TLR4) was significantly increased, which was associated with the upregulation of interleukin (IL)­1ß and tumor necrosis factor (TNF)­α expression. Furthermore, overexpression of miR­199a­3p significantly suppressed the expression and activation of HMGB1 and TLR4/NF­κB signaling, and decreased the levels of IL­1ß and TNF­α in vitro and in vivo. Additionally, overexpression of HMGB1 and/or TLR4 reversed the anti­inflammatory effects of miR­199a­3p mimics in vitro and in vivo. These results indicate that miR­199a­3p acts as a negative inflammatory regulator in PTB by targeting HMGB1 to regulate the TLR4/NF­κB pathway.

Benzene polycarboxylic acid - A useful marker for condensed organic matter, but not for only pyrogenic black carbon.

Benzene polycarboxylic acid (BPCA) molecules are a widely used marker method for the qualitative and quantitative analysis of pyrogenic black carbons (BC). Based on an overview of the development and chemical reaction mechanism of the BPCA method, we propose that the commonly used BPCA markers may not be solely indicative of BC but more generally of condensed organic matter in soils and aquatic systems. First, we sequentially removed the soil fractions and observed that the BPCA contents were abundant in humic acids (HAs). After sequential treatment, the residual particles were supposed to contain BC and minerals; however, the BPCAs in the residue accounted for only 2.4-10.1% of that detected in the entire soil. In addition, substantial quantities of BPCAs were detected in both thermally treated samples and composted biomass. Furthermore, humic acids extracted from all the samples showed that obvious BPCA contents in the samples accounted for 0.1-121.7 mg/g. Therefore, soil fractionation may also partly extract BCs as suggested by BPCAs in the HAs of the biochars. However, organic matter without any thermal treatment may contain BPCAs. A series of standard substances without any BC showed high BPCA content in the samples from 5.9-124.5 mg/g. These observations create a serious concern for the proper application of BPCAs as a marker for BCs. Combining a systematic literature review of BPCA that deviates from BC content, we suggest that the BC-like structure of organic matter may be referred to as BPCA-probed organic matter content, which could be a more useful term for studies on the multimedia environmental behaviors of contaminants.

Transfer of polycyclic aromatic hydrocarbons from mother to fetus in relation to pregnancy complications.

The accumulation and transfer of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), in the human body, especially from mother to fetus, has been the subject of many research studies, but the related data are limited and the mechanisms are unknown. This is the first study to investigate the distribution of PAHs in paired samples of maternal blood, placenta tissue, and umbilical cord blood in relation to pregnancy complications. Sixty-four pairs of samples were collected in Kunming, China; 18 were from healthy pregnant women and 46 were from patients with pregnancy complications. The predominant PAHs in these pregnant women were high-molecular-weight (HMW) compounds, mainly from the incomplete combustion or pyrolysis of biomass. In the control group, the total amount of HMW compounds (ΣHMWPAHs) was significantly higher in maternal blood than in umbilical cord blood, which suggested that placenta may decrease PAH transfer in healthy pregnant women. However, this phenomenon was not observed for low-molecular-weight PAHs or in the case group. In the control group, Σ16PAH and ΣHMWPAH in the placenta were higher than those in maternal blood and umbilical cord blood; for the case group, a contrasting result was observed. ΣHMWPAHs in the placenta was significantly higher in the control group than in the case group. The same results were observed after the exclusion of the impact of the genotypes of the PAH metabolic enzymes (both phase I and phase II enzymes). Thus, the decreased PAH transfer from mother to fetus may partially result from the accumulation of PAHs inside the placenta.

Molecular markers of benzene polycarboxylic acids in describing biochar physiochemical properties and sorption characteristics.

Biochar function in soil is based on properties such as sorption characteristics, and these are expected to change throughout the life cycle of the biochar. Because biochar particles cannot easily be separated from soil particles, this change is seldom investigated. Biochar-related molecular markers, such as benzene polycarboxylic acids (BPCAs) are promising tools for studying the properties of biochars in complex environmental matrices. In this study, biochars were derived from corn straw and pine wood sawdust at 200-500 °C, and their aging was simulated with NaClO. Biochar properties were characterized by elemental analysis, BET surface characterization and BPCA molecular marker analysis. Chemical oxidation decreased the surface area (SA) but increased the O content of biochars. The oxidation decreased the amount of biochars, with a mass loss in the range of 10-55%. A similar mass loss was also observed for BPCAs and was negatively related to both the pyrolysis temperature and the extent of the condensed structure (higher aromaticity). The biochar amounts were calculated quantitatively using the sum of BPCA contents, with a conversion factor (the ratio of biochar amount to BPCA content) in the range of 3.3-5.5, and were negatively related to the B5CA content. Three model pollutants, namely, bisphenol A (BPA), sulfamethoxazole (SMX), and phenanthrene (PHE), were chosen to study the sorption characteristics of biochar before and after oxidation. Chemical oxidation generally increased SMX sorption but decreased PHE sorption. The nonlinear factor n, based on Freundlich equation modeling, was negatively related to B6CA for all three chemicals. The BPCA molecular markers, especially B5CA and B6CA, were correlated to the biochar properties before and after oxidation and are thus a potentially useful technique for describing the characteristics of biochar in the environment.

The Overlooked Occurrence of Environmentally Persistent Free Radicals in an Area with Low-Rank Coal Burning, Xuanwei, China.

The mining and burning of low-rank coal in Xuanwei, China have attracted a great deal of research attention because of the generated polyaromatic hydrocarbons (PAHs) and the high incidence of lung cancer in this region. Given the abundant transition metals in the allitic soil, we hypothesized that environmentally persistent free radicals (EPFRs) are formed in this region and the potential risk had not been addressed. Strong electron paramagnetic resonance (EPR) signals of 3.20 × 1017 - 3.10 × 1019 spins/g were detected in environmental samples, including chimney soot, coal, soil and total suspended particles (TSP). These EPR signals did not significantly change after 18-months storage and had g-values in the range of 2.0039-2.0046, suggesting typical organic free radicals. Similar strong EPR signals were observed in PAH (anthracene and pyrene as model compounds) degradation on simulated soil particles and lasted over one month even when the applied PAHs were 100% degraded. Based on g-value and bond width, we propose that EPR signals detected in TSP and soot originated from both coal combustion and PAH photodegradation. Further research is thus urgently required to investigate EPFR generation, exposure and risk in Xuanwei to better understand the cause of high lung cancer incidence.

Increased Expression of Thioredoxin-1 Binding Protein-2 in Placentas in Preeclampsia.

OBJECTIVE: To examine the expressions of thioredoxin-1 (TRX-1) and thioredoxin-1 binding protein-2 (TBP-2) in placentas affected by preeclampsia. STUDY DESIGN: We examined the mRNA levels of TRX-1, TBP-2, cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) in preeclamptic (n=20) and normal placentas (n=18) by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: We found the mRNA level of TRX-1 was significantly decreased (p<0.005), while the mRNA levels of TBP-2, COX-2, and TNF-α were significantly increased in the placentas in preeclampsia when compared to the normal group (p<0.005). CONCLUSION: These data suggest that TBP-2 may play roles in the pathophysiology of preeclampsia, probably by contributing to oxidative stress and inflammation. Thus, TBP-2 may be a potential therapeutic target for preeclampsia.

Expression of 5-HT3 receptors and TTX resistant sodium channels (Na(V)1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders.

BACKGROUND: Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. METHODS: Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. RESULTS: A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly higher in patients (42.6%) compared to healthy controls (12.0%) (P < .001). CONCLUSIONS: This study showed that the 5-HT3A-receptor is highly expressed in human masseter and tibialis muscles and that there are more nerve fibers that express 5-HT3A-receptors in the masseter of women with myofascial TMD compared to healthy women. These findings indicate that 5-HT3-receptors might be up-regulated in myofascial TMD and could serve as potential biomarkers of chronic muscle pain.

Nerve growth factor alters the sensitivity of rat masseter muscle mechanoreceptors to NMDA receptor activation.

Intramuscular injection of nerve growth factor (NGF) into rat masseter muscle induces a local mechanical sensitization that is greater in female than in male rats. The duration of NGF-induced sensitization in male and female rats was associated with an increase in peripheral N-methyl-d-aspartate (NMDA) receptor expression by masseter muscle afferent fibers that began 3 days postinjection. Here, we investigated the functional consequences of increased NMDA expression on the response properties of masseter muscle mechanoreceptors. In vivo extracellular single-unit electrophysiological recordings of trigeminal ganglion neurons innervating the masseter muscle were performed in anesthetized rats 3 days after NGF injection (25 µg/ml, 10 µl) into the masseter muscle. Mechanical activation threshold was assessed before and after intramuscular injection of NMDA. NMDA injection induced mechanical sensitization in both sexes that was increased significantly following NGF injection in the male rats but not in the female rats. However, in female but not male rats, further examination found that preadministration of NGF induced a greater sensitization in slow Aδ-fibers (2-7 m/s) than fast Aδ-fibers (7-12 m/s). This suggests that preadministration of NGF had a different effect on slowly conducting mechanoreceptors in the female rats compared with the male rats. Although previous studies have found an association between estrogenic tone and NMDA activity, no correlation was observed between NMDA-evoked mechanical sensitization and plasma estrogen level. This study suggests NGF alters NMDA-induced mechanical sensitization in the peripheral endings of masseter mechanoreceptors in a sexually dimorphic manner.

The expression of thioredoxin-1 in preterm delivery placenta.

UNLABELLED: Preterm delivery (PTD) is the leading cause of infant mortality and morbidity. However, the mechanism at the molecular level is still unknown. Placental inflammatory response and oxidative stress are associated with PTD. Thioredoxin-1 (TRX-1) regulates oxidative stress, inflammation, and the activities of transcription factors. OBJECTIVES: The objective was to detect in placental tissues the expressions of TRX-1 and the TRX-1-related molecules: tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), thioredoxin-1-binding protein-2 (TBP-2), hypoxia inducible transcription factor 1α (HIF-1α), and forkhead box protein O3A (FoxO3A). METHODS: PTD was defined as gestation of <37 weeks and term delivery (TD) as ≥37 weeks. The expressions of TRX-1 and TRX-1-related molecules were examined in placental tissues by real-time polymerase chain rection and western blot. RESULTS: The expressions of TRX-1, TNF-α, COX-2, HIF-1α, and FoxO3A in the placenta of PTD were significantly higher as compared with TD, but no difference was observed in TBP-2 expression. DISCUSSION: These results indicate that TRX-1 may be adaptively induced by the effects of inflammation and oxidative stress, suggesting protective roles for TRX-1 against these effects in the placenta of PTD.

Intramuscular ketorolac inhibits activation of rat peripheral NMDA receptors.

The nonsteroidal anti-inflammatory drug (NSAID) diclofenac has local anesthetic-like and peripheral N-methyl-d-aspartate (NMDA) receptor antagonist characteristics when administered at higher concentrations to masticatory muscle. It is not known if the ability to inhibit NMDA receptors is unique to diclofenac or shared by other NSAIDs. This study was undertaken to determine whether intramuscular injection of ketorolac or naproxen at concentrations that do not induce local anesthetic-like effects could attenuate jaw-closer muscle nociceptor discharge in anesthetized Sprague-Dawley rats. It was found that ketorolac (5 mM) inhibited hypertonic saline-evoked nociceptor discharge, which suggests that at this concentration, ketorolac has local anesthetic-like properties. A lower concentration of ketorolac (0.5 mM), which did not affect hypertonic saline-evoked discharge, did inhibit nociceptor discharge evoked by NMDA. In contrast, naproxen (5 mM) did not alter hypertonic saline- or NMDA-evoked nociceptor discharge. Subsequent experiments revealed that ketorolac (0.5 mM) had no effect on nociceptor discharge evoked by αß-methylene ATP, 5-hydroxytryptamine, or AMPA. The inhibitory effect of ketorolac did not appear to be related to cyclooxygenase inhibition, because the concentration of prostaglandin E(2) in the masticatory muscles 10 min after injection of either NSAID was not significantly decreased. The present study indicates that in vivo, ketorolac, but not naproxen, can antagonize NMDA-evoked nociceptor discharge similarly to diclofenac. We speculate that structural similarities between ketorolac and diclofenac could account for the ability of these NSAIDs to inhibit NMDA-evoked nociceptor discharge. These properties may partly explain the analgesic effect of intramuscularly injected ketorolac in the clinic.

TNFα mechanically sensitizes masseter muscle nociceptors by increasing prostaglandin E2 levels.

TNFα induces mechanical sensitization of rat masseter muscle nociceptors, which takes 2-3 h to manifest and is mediated through activation of P55 and P75 receptors. This study was undertaken to determine whether TNFα induces nociceptor mechanical sensitization through the release of other algogenic substances such as glutamate, prostaglandin E(2) (PGE(2)), and/or nerve growth factor (NGF), which have been shown to induce mechanical sensitization of muscle nociceptors. Masseter muscle homogenate levels of PGE(2) and NGF were measured 3 h after injection of TNFα (1 µg) or vehicle control using commercially available kits. Interstitial glutamate concentration was measured after injection of TNFα or vehicle control using a glutamate-selective biosensor probe. Diclofenac, a cyclooxygenase inhibitor that blocks the synthesis of PGE(2), D-2-amino-5-phosphonovaleric acid (APV), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and a tyrosine kinase A (TrkA) receptor antibody, which blocks NGF-induced masseter muscle nociceptor sensitization, were used to assess the contribution of PGE(2), glutamate, and NGF to TNFα-induced nociceptor sensitization. PGE(2) and glutamate concentrations were significantly elevated 3 h after TNFα injection into the masseter muscle. Injection of diclofenac partially reversed the TNFα-induced decreases in the mechanical threshold (MT) of masseter muscle nociceptors, whereas vehicle control, APV, and TrkA antibody did not significantly alter nociceptor MT. These results suggest that TNFα-induced mechanical sensitization of masseter muscle nociceptors is mediated in part by increased PGE(2) levels. The findings of this study support the hypothesis that TNFα induces a delayed mechanical sensitization of masseter muscle nociceptors indirectly by the release of PGE(2).

Sensitization of rat facial cutaneous mechanoreceptors by activation of peripheral N-methyl-d-aspartate receptors.

The effect of subcutaneous injection of glutamate on the mechanical sensitivity of rat facial cutaneous mechanoreceptors was examined. Individual facial mechanoreceptors were recorded in the trigeminal ganglion of anesthetized Sprague-Dawley rats. An electronic von Frey hair was used to measure the mechanical threshold (MT) of the afferent fibers at baseline and following subcutaneous injection of glutamate (0, 0.01, 0.1, 1M; 10microl) or glutamate (0, 0.1M) plus the competitive N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV; 0.01M). Subcutaneous injections were randomized and the investigator was unaware of their content. Changes in MT were assessed with a repeated measure ANOVA with time, sex and treatment as factors. Immunohistochemistry was used to confirm NMDA receptor expression by cutaneous nerve fibers. A total of 100 (50 per sex) facial mechanoreceptors were recorded from 61 (32 females, 29 males) rats in two separate experiments. Subcutaneous injections of higher concentrations of glutamate (1, 0.1M) induced a significant mechanical sensitization of skin afferent fibers (compared to 0 and 0.01M). Females (EC(50)=16.2mM) were more sensitive to glutamate than males (EC(50)=73.0mM). Facial cutaneous nerve fibers in both sexes expressed NMDA receptors. APV blocked the mechanical sensitization of the afferent fibers treated by glutamate 0.1M in both sexes with a lower effect in females at a 10-20minute post-injection. Subcutaneous injection of glutamate mechanically sensitizes rat facial cutaneous mechanoreceptors through activation of peripheral NMDA receptors. Peripheral NMDA receptor antagonists may be considered for craniofacial pain.

Human nerve growth factor sensitizes masseter muscle nociceptors in female rats.

Injection of nerve growth factor (NGF) into the masseter muscle is not painful but does induce a localized, quick onset ( approximately 1h) and long-lasting mechanical sensitization in healthy human subjects. We tested the hypothesis that human NGF mechanically sensitizes masseter muscle nociceptors by increasing the sensitivity of peripheral N-methyl-d-aspartate (NMDA) receptors. Co-expression of the NR2B subunit of the NMDA receptor with P75 and TrkA NGF receptors by trigeminal ganglion neurons that innervate the masseter muscle was investigated immunohistochemically. Nociceptor activity was recorded extracellularly from the trigeminal ganglion of anaesthetized female rats. Nociceptor mechanical threshold was assessed before and every 30 min for 3h after injection of human NGF (25 microg/ml, 10 microl, n=12), and in subsequent experiments NGF with TrkA (n=12) or P75 (n=11) receptor antibodies. Glutamate (1M, 10 microl) was injected at the end of each experiment. Approximately 85% of NR2B positive masseter ganglion neurons co-expressed P75 or TrkA receptors, suggesting the potential for interaction. When compared with the vehicle control, it was found that injection of NGF into the masseter muscle did not evoke significant nociceptor discharge but did significantly reduce nociceptor mechanical threshold ( approximately 30%). There was no effect of NGF on glutamate-evoked nociceptor discharge or glutamate-induced mechanical sensitization. Additional experiments indicated that NGF-induced mechanical sensitization could be partially attenuated with TrkA receptor antibodies, but not P75 receptor antibodies. These findings indicate that human NGF-induced sensitization of masseter nociceptors results, in part, from the activation of TrkA receptors, but does not appear to be mediated through enhanced peripheral NMDA receptor activity.

TNFalpha mechanically sensitizes masseter muscle afferent fibers of male rats.

Behavioral evidence in rats indicates that injection of tumor necrosis factor alpha (TNFalpha) into skeletal muscle results in a prolonged mechanical sensitization without gross inflammation. To investigate whether a peripheral mechanism could underlie this effect, in the present study, TNFalpha (1 or 0.1 microg) was injected into the rat masseter muscle to assess its effect on the excitability and mechanical threshold (MT) of muscle nociceptors as well as on inflammation. Expression of TNFR1 (P55 receptors) and TNFR2 (P75 receptors) by the masseter muscle and trigeminal ganglion neurons that innervate that muscle was determined by Western blot and immunohistochemistry, respectively. The Evans blue dye technique was used at the end of the TNFalpha experiments to assess for plasma protein extravasation. In subsequent experiments to confirm the involvement of receptor activation in TNFalpha-induced effects, P55 or P75 receptor antibody was co-injected with TNFalpha. Intramuscular injection of 1 microg TNFalpha did not excite nociceptors but did significantly decrease MT compared with vehicle control. There was no evidence of gross inflammation 3 h after injection of TNFalpha. Co-injection of TNFalpha with P55 or P75 receptor antibodies attenuated TNFalpha-induced mechanical sensitization. P55 and P75 receptors were expressed by 29 and 62% of masseter nociceptors, respectively. These findings indicate that TNFalpha induces mechanical sensitization of masseter nociceptors that is mediated through activation of peripheral P55 and P75 receptors. These results support the hypothesis that a peripheral receptor mechanism could contribute to TNFalpha-induced noninflammatory mechanical sensitization of skeletal muscle previously reported in behaving rats.