The purpose of this study was to demonstrate that daily 40-minute whole body exposure to an inhomogeneous static magnetic field (SMF) prolongs induced preterm birth (PTB) in mice.
The murine model for PTB induction was performed by the administration of 25 μg/animal lipopolysaccharide (LPS) intraperitoneally. The applied SMF was an inhomogeneous gradient field with 2.8-476.7 millitesla peak-to-peak magnetic induction range by 10 mm lateral periodicity. During SMF exposure, mice were free to move in their cage.
The fetal development and the delivery were normal in animals that were exposed to SMF but not treated with LPS. SMF in these cases did not influence the term of delivery. In LPS-challenged animals, SMF exposure prolonged the time of PTB occurrence from 17.43 h (n = 7) to 21.93 h (n = 15) after the challenge ( P < .05).
Exposure to inhomogeneous SMF may have a valuable effect in the prevention of PTB and may have clinical relevance to humans.
Despite intensive research, preterm birth (PTB) remains a central problem in modern perinatal medicine. PTB occurs in 5-10% of all pregnancies. Clinical, epidemiologic, and experimental studies indicate that urogenital tract infections play a critical role in the pathogenesis of PTB. Moreover, intrauterine infections often are associated with the development of organ system diseases, such as cerebral palsy, asphyxia, or chronic lung disease. They significantly increase perinatal morbidity and mortality rates. Ascending infection occurs when pathogenic bacteria pass the cervical barrier. They can cause decidual and chorioamnion inflammation that is characterized by the bacterial infection of the amniotic fluid. The presence of bacteria induces the release of proinflammatory cytokines (ie, interleukin [IL]-1 and tumor necrosis factor) by macrophages, amnion, deciduae, and myometrium. These pathophysiologic mechanisms are leading to the induction of labor and dilation of the cervix ; moreover, cytokines play a role in balancing between neurotrophism and neurotoxicity.
The wide spectrum of reasons that lead to PTB has not been clarified yet. In the past decades, treatment and management of PTB have not improved much; the rate of PTB occurrence did not change significantly. It has been known for a long time that infections in mothers raise the risk of complications that occur during delivery. Several risk factors of PTB have been identified: intrauterine infection, inflammation, vascular pathophysiologic conditions, uteroplacental ischemia, intrauterine bleeding, and overtension of the uterus. Genetic polymorphisms that enhance the intensity and/or the duration of the inflammatory reaction may also increase the risk of PTB.
Several of the well-known risk factors of PTB increases inflammatory disposition. It is well-known that inflammatory processes trigger a wide range of uterotonic factors (eg, the influx of inflammatory cells into the uterus). Elevated levels of proinflammatory cytokines (tumor necrosis factor–α, IL-1β, IL-6, IL-8) and prostaglandins have been observed in the human amnion during parturition. Proinflammatory cytokines can induce preterm spasms of the pregnant uterus by synthetizing prostaglandins among others. The physiologic activation of the decidua is responsible for the induction of uterine contractions. Latent inflammation in the uterus can activate the decidua well before term, which may lead to preterm contractions and to PTB. The risk of PTB under autoimmune conditions is high, which suggests the participation of inflammatory processes that are mediated by the immune system in the commencement of preterm spasms of the uterus.
In the present experiments, we used a murine model for PTB to explore the relation of inflammation of the uterus and PTB. Either local or systemic exposure to microbial products led to PTB in several experimental models. Lipopolysaccharide (LPS) plays a key role in eliciting an inflammatory response that includes the activation of the immune cells and the release of enzymes that are involved in the remodeling of the extracellular matrix that leads to PTB. LPS is recognized by a protein of the innate immune system that includes Toll-like receptor 4, which serves as a sentinel of uterine immunity in the context of response to pathogens. The connection between PTB and bacterial inflammation is proved by the effective antibiotics therapy in women at risk of PTB.
There are reports of experimental studies that have shown that rodents that are exposed to a static magnetic field (SMF) exhibited strong analgesia to various painful stimuli. SMF exposure has been demonstrated to deaden pain that originates from the inflamed peritoneum in the mouse.
The direct effect of SMF exposure on inflammatory conditions was studied by Morris and Skalak. Localized inflammation was induced by the injection of inflammatory agents lambda-carrageenan or histamine into rat hindpaws, alone or in conjunction with pharmacologic agents. Application of a 10 or 70 millitesla (mT), but not 400 mT of SMF for 15 or 30 min immediately after histamine-induced edema resulted in a significant, 20-50% reduction in edema formation. In addition, a 2 h 70 mT SMF exposure to λ-carrageenan–induced edema also resulted in significant (33-37%) edema reduction. In another study, inhomogeneous SMF exposure significantly diminished the duration of formalin-evoked paw lickings and liftings in both phase I (acute somatic nociception) and phase II (acute inflammatory nociception). Selective inactivation of capsaicin-sensitive sensory fibers by high-dose resiniferatoxin pretreatment decreased nocifensive behaviors in phase II of the formalin test. This suggested that proinflammatory neuropeptides (such as substance P and calcitonin gene-related peptide that were released from these fibers) were involved in this inflammatory reaction.
Human experiences with local SMF exposure on the pelvic domain are rare. Holcomb et al studied 2 patients with a diagnosis of intervertebral disk disease at spinal cord levels that could be responsible for their chronic pain in the abdomen and genitals. The authors found a surprisingly rapid relief in both cases on continuous local SMF exposure with a quadrupolar arrangement of 4 permanent magnets ( B r = 1.2 T); the effect was sustained for over 2 years. Brown et al performed a randomized, double-blind, placebo-controlled trial with 32 patients at a gynecology clinic. They confirmed a significant effect in Pain Disability Index, Clinical Global Impressions-Severity, and Clinical Global Impressions-Improvement scores between the group that had been treated with continuous local exposure with 50 mT and the group that received sham exposure. They admitted though that patients could likely identify their treatment.
The main initiative to perform our experiments was potentially to widen the treatment options of pregnant women with pain and inflammation. Many of the currently available clinical treatments of the inflammatory state have limited effectiveness, and their action is often accompanied by distressing side-effects. The search for reliable, safe, and effective treatments for inflammation remains a major medical problem; not surprisingly, patients continuously have been exploring complementary or alternative approaches. Among a number of other treatment strategies, magnetic therapy is increasingly used to alleviate pain and shorten the period of healing. Magnets appeal to patients because they promise a simple solution for pain relief and are relatively safe, drug free, durable, and noninvasive. The proliferation of high-field magnetic resonance imaging (MRI) equipment makes it necessary to deal with any possible (beneficial or adverse) effects of SMF, gradient magnetic field and radiofrequency electromagnetic radiation that may be exerted on living tissues, with special emphasis on pregnant women. We decided to test the effectiveness of SMF exposure in inflammation-induced PTB first in the literature. Inflammation was produced by intraperitoneally administered bacterial LPS in mice. The rationale was to explore the effect of an inhomogeneous SMF exposure on inflammation-induced PTB in a widely used experimental rodent model under easily reproducible experimental circumstances with an SMF that was optimized for pain inhibition and validated for rodent experiments. We also planned to make dose dependence, if possible.
Materials and Methods
Inhomogeneous SMF was induced with an exposure system that was developed, validated, and optimized for animal experiments by László et al. The device consisted of 2 ferrous matrices that contained 10 × 10 mm (diameter × height) cylindric neodynium-iron-boron N50 grade magnets ( B r = 1.47 T). The lateral periodicity of the inhomogeneous SMF was 10 mm. The individual magnets (ChenYang Technologies GmbH & Co KG, Finsing, Germany) in both matrices were placed next to each other with alternating polarity. Magnets that were facing each other in the 2 matrices were oriented with opposite polarity. The matrices were fixed in a holder in which the matrices were separated from each other by a distance of 50 mm, thus creating an exposure chamber. Magnetic coupling was applied between the matrices. This arrangement allowed us to insert a 140 × 140 × 46 mm (length × width × height) perforated plexiglas animal cage with air holes into the exposure chamber. An air-permeable opaque material covered the cage on 4 sides to make illumination circumstances similar in the exposure chamber and in the sham experiment. The generator and the cage can be seen in Gyires et al.
Dosimetry was performed separately from the animal experiments by means of a 5 V calibrated ratiometric linear Hall-effect sensor of 12.3 mV/T sensitivity (model UGN3503; Allegro MicroSystems, Inc, Worcester, MA). The typical peak-to-peak magnetic induction values along the axis of a magnet in the isocenter were 476.7 ± 0.1, 12.0 ± 0.1, and 2.8 ± 0.1 mT, whereas the average lateral gradient values between 2 neighboring local extremes were 47.7, 1.2, and 0.3 T/m at 3, 15, and 25 mm from the surfaces of matrices, respectively. This description of the SMF complies with the requirements that Colbert et al proposed for standardization.
The 40-minute time period of SMF exposure was chosen to obtain results comparable with those in the writhing test and in the neuropathy model. The idea of the frequency of SMF exposure originates from the literature and was confirmed by László et al ; robust results could be achieved with the same magnetic treatment under different, induced pathologic conditions. SMF exposure that was created with this specific magnetic exposure system was shown not to introduce significant changes in the behavior of animals.
Escherichia coli LPS (serotype 0111:B4; Sigma-Aldrich, Budapest, Hungary) was dissolved in phosphate buffer solution. LPS 25 μg/mouse (approximately 1 mg/kg) was administered intraperitoneally in the mice on a single occasion on day 15 of gestation. According to the literature, this dose induces PTB within 17 h after the challenge.
Timed-pregnant C57BL/6 mice (Charles-River Laboratories, Isaszeg, Hungary) were used. Gestation age was determined by the presence of a vaginal plug (day 0 of gestation). The conventional timing of gestation was used; pregnancy begins with the maternal recognition of the embryo around the time of implantation (day 5 of gestation).
Thirty-four female mice were divided randomly into the following 5 experimental groups: group 1 comprised intact (untreated) gravid animals (n = 6) and was the negative control group; expected delivery was on day 19 of gestation; group 2 comprised gravid animals that were exposed to 40 minutes per day (min/d) whole body SMF exposure starting on day 14 of gestation (cumulated exposure time, 6 × 40 min; n = 6); expected delivery was on day 19 of gestation; group 3 comprised gravid animals that were not exposed to SMF (n = 7) and served as the sham (positive) control group; animals were treated with intraperitoneal LPS to induce PTB; group 4 comprised gravid animals that were exposed to 40 min/d whole body SMF exposure starting on day 14 of gestation until delivery (cumulated exposure time 3 × 40 min, n = 7); animals were treated with intraperitoneal LPS to induce PTB; group 5 comprised gravid animals that were exposed to 40 min/d whole body SMF exposure starting on day 1 of gestation until delivery (cumulated exposure time, 17 × 40 min; n = 8).
One male mouse was allocated randomly to each group of females. These male mice were used for impregnating all the females in their specific groups and were then separated from the females. Male mice did not participate in the experiment otherwise; they were neither injected with LPS, nor were they exposed to SMF.
Control animals (both negative and sham) were kept for 40 min/d in identical perforated plexiglas boxes. For all treatments (sham and SMF), we put 2 animals into the plexiglas cage at a time, keeping in mind that mice are socially sensitive ; then, the cage with the animals was either inserted into the exposure chamber (SMF) or left alone (sham) for 40 min every day of treatment.
The treatment of animals in groups 2 and 4 started on gestation day 14 only, because previous experimental results suggested that the SMF treatment of healthy animals may not have any physiologic effects. However, we tested this assumption by introducing group 5. We presumed that data of groups 4 and 5 could be pooled, if their averages did not significantly differ from each other. The flow diagram of the experimental series can be seen in Figure 1 .