Intrapartum fever complicates 20%–33% of deliveries in women with labor epidural analgesia, an incidence several times higher than that of women laboring without epidurals.1 The phenomenon has been noted in observational studies, before/after (sentinel event or natural experiment) analyses, and randomized controlled trials. The bulk of evidence supports an inflammatory but noninfectious etiology of this excess fever.1 Because >60% of women in the United States deliver with epidural analgesia,2 this represents a significant incidence of excess fever. Maternal intrapartum fever leads to increased antibiotic exposure3 and cesarean delivery4 in women, and may have profound negative effects on newborns, most importantly, including the developing fetal brain.5–8
While there is no comprehensive animal model of labor epidural analgesia, there are models of maternal infectious inflammation demonstrating fetal or neonatal brain injury.9 However, given that epidural-associated fever is inflammatory but noninfectious, we attempted to model the clinical situation seen in human labor epidural analgesia related to fever by inducing noninfectious inflammatory fever in the near-term pregnant rat and investigated the effects on the fetal brain. A common finding in epidural-associated fever, as well as human epidemiologic investigations linking maternal fever to neonatal brain injury, is elevated interleukin (IL)-6, a proinflammatory cytokine.10,11 IL-6 levels increase with increasing exposure to epidural analgesia10 and are higher in women developing fever than in those remaining afebrile.12 Maneuvers such as maternal corticosteroid administration, which reduces IL-6 levels, also block maternal epidural-associated fever,13 whereas other approaches that do not directly target inflammatory mediators, such as acetaminophen14 or antibiotics,15 do not.
Therefore, we developed a model of noninfectious inflammatory fever in the near-term pregnant rat using physiologic IL-6 levels comparable to those encountered in human patients with labor epidural analgesia. We hypothesized that brief administration of IL-6 would induce an increase in core temperature in the dams, and secondarily that maternal IL-6 would induce neuroinflammation in the fetus.
All procedures in this investigation were in accordance with the Guide for the Care and Use of Laboratory Animals16 and the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines.17 After obtaining approval from the Animal Care and Use Committee at Tufts University School of Medicine and the Sackler School of Biomedical Sciences Department of Laboratory and Animal Medicine, 24 near-term pregnant Harlan Sprague-Dawley rats, weighing 285–325 g at the time of experiments, were studied between September 2013 and August 2014. A total of 32 fetal rats were subsequently studied, as detailed below: 12 for ED-1 staining, 12 for cyclooxygenase-2 (COX-2) staining, and 8 for COX-2 Western blotting analysis. Animals were housed at 22°C and under a 12-hour light-dark cycle, with free access to food and water. All experiments were performed on gestational day 20 (term pregnancy: ≈22 days).
All cesarean deliveries were performed after deep anesthesia with intramuscular 10 and 100 mg/kg of xylazine and ketamine, respectively.
Fetuses were delivered within 1 minute from mothers’ anesthetic. Newborns were killed by decapitation, and heads were frozen immediately in dry ice or immersed in 4% paraformaldehyde fixative. The maternal chest was opened, and maternal blood was sampled by puncturing the left ventricle under direct vision. At the conclusion of sampling, the animals were killed by exsanguination.
The study was divided into 2 parts. The first part included serial drug injections, maternal temperature measurements, and IL-6 maternal blood sampling. The second part comprised neonatal brain analysis.
Drug Administration, Temperature Measurements, and IL-6 Maternal Plasma Levels.
On gestational day 20, pregnant rats were moved from the animal facility to the laboratory and allowed 1-hour acclimation before temperature testing. Two groups of near-term pregnant rats (n = 12 per group) were randomized from a computer-generated randomization scheme (SAS Institute, Cary, NC) to receive either of the following: (1) 3 serial vehicle intramuscular injections every 90 minutes; or (2) 3 serial rat recombinant IL-6 intramuscular injections in the gluteal region (every 90 minutes, 1, 1.5, and 2.5 μg, respectively). The increasing dose protocol was chosen to mimic the clinical observation of increasing IL-6 with longer exposure to epidural analgesia.10 All animals received the injections at the same time of day. Baseline temperatures were taken immediately before the first injection (Microtherma 2 digital rectal thermometer; ThermoWorks, Alpine, UT). After the first injection, serial core rectal temperature measurements were intermittently taken from pregnant dams every 30 minutes until the end of the experiment. Animals underwent cesarean delivery and were killed 8 hours after the first treatment. Neonatal weights were quickly recorded before decapitation.
While pregnant rats were deeply anesthetized, the chest was opened, and 5–10 mL of blood was aspirated from the left ventricle through a 20-gauge needle. After collection of the whole blood, the blood was allowed to clot by leaving it undisturbed at room temperature for 30 minutes. The clot was then removed by centrifuging at 2000g for 15 minutes in a refrigerated centrifuge. Serum was apportioned into 0.5-mL aliquots and stored at –80°C. At the time of analysis, serum was thawed and assayed randomly from each treatment group (n = 4 per group) for IL-6 using a commercially available IL-6 rat Enzyme-Linked Immunosorbent Assay (ELISA; Abcam, Cambridge, MA). The assay used an antibody specific for rat IL-6 coated on a 96-well plate. Assay runs were in triplicates for each serum sample. Standards and samples were pipetted into the wells, allowing IL-6 present in a sample to bind to the immobilized antibody. The wells were washed, biotinylated anti-rat IL-6 antibody was added and incubated, and wells were rinsed to remove the unbound biotinylated antibody. Horseradish peroxidase (HRP)-conjugated streptavidin was added and incubated, and then wells were rinsed, and tetramethylbenzadine substrate solution was added, generating a color reaction proportional to the amount of IL-6 bound. The intensity of the absorbance was measured at 450 nm, and IL-6 concentration levels were expressed in picogram per milliliter.
Neonatal Brain Analysis.
ED-1/CD68 is a marker of activated microglia. Brains were immersed in fixative at 4°C overnight and were transferred to 30% sucrose at 4°C overnight. To enable identical processing across groups of neonatal brains, 12 brains (n = 6 saline, n = 6 IL-6) were embedded in 7% gelatin/30% sucrose and immersed in fixative overnight. Gelatin-embedded brain blocks were frozen and 33 µm sections cut on a sliding microtome from the level of the rostral septal nuclei to the caudal hippocampus. We chose these brain regions because of their importance in learning and memory functions and other work suggesting damage to these areas was potentially functionally important.18 Sections were mounted on slides, dried overnight at room temperature, and stored and desiccated at −20°C, until assay. For detecting activated microglia, a mouse monoclonal antibody specific to ED-1/CD68 protein (Millipore, Temecula, CA) was used at 1:2000. Slide-mounted sections were incubated with the CD68 antibody at 4°C overnight, rinsed and incubated with Impress mouse-AP polymer (Vector, Burlingame, CA) followed by substrate reaction using Vector Blue (Vector). For each neonatal brain, images of ED-1–labeled cells were acquired at 2 levels (200 μm interval) for the following 2 brain regions: lateral septum and caudal hippocampus. Quantitation of ED-1 cells was performed by detection of a blue ED-1 color reaction. Sections were viewed with a Zeiss microscope at ×10 and photographed using a Canon digital microscope (Canon USA, Huntington, NY), with 21 megapixel resolution. The number of pixels occupied by ED-1 cells was measured automatically using image analysis software (SigmaScan; Jandel Scientific Inc, San Rafael, CA). All sections were processed simultaneously using the same brightness and contrast settings and threshold for image analysis.
COX-2 Enzyme Immunohistochemistry and Western Blotting.
COX-2 is a marker of neuroinflammation. From each of 6 vehicle-injected and 6 IL-6–injected dams, fetal brains were divided into 2 groups (n = 6 per group) according to whether the dams received saline or IL-6. For immunohistochemistry of COX-2 expression, 33 µm sections from gelatin brain blocks described above were incubated with primary antibody rabbit anti-COX-2, 1:800 (Novus Biological, Littleton, CO) overnight at 4°C, followed by secondary antibody ImmPRESS anti-rabbit IgG HRP (Vector) and then reacted with the substrate Nova Red (Vector).
Images of the periventricular regions of the brain were captured at ×4 magnification using a CoolSnap digital camera (Roper Scientific, Tucson, AZ). Quantification of immunostaining was performed on 5 randomly selected sections from the rostral to caudal septum for each animal. Image analysis software (Image J; NIH Image; National Institutes of Health, Bethesda, MD) was used to quantify COX-2 immunoreactive areas. The upper and lower threshold optical densities were adjusted to match positive immunoreactivity. The image thresholds were determined and applied uniformly to all sections. A fixed area (1.6 × 2 mm2) was positioned at the level of the lateral septum to include all regions surrounding the lateral ventricle (topographically corresponding to the subventricular zone [SVZ]), and the number of pixels within the threshold value was quantified. Data are expressed as number of pixels in the area. To limit variability in immunohistochemical measurements, all groups of animals were processed on the same day and the same threshold value was applied to all images for a given antibody. The individual who quantified the images was blind to the treatment group. To avoid experimenter bias, auto-adjustment of brightness and contrast, as well as threshold of staining signal, was performed by National Institutes of Health software.
Eight fetuses were analyzed for forebrain COX-2 quantitation by Western blotting. We again chose those brain areas, such as the nucleus caudatus, given their role played in learning processes19 and the prominence in the staining for COX-2 in the SVZ. Brains were divided into 2 groups (n = 4 per group) according to whether the dams received saline or IL-6. Neonatal forebrains that were frozen at killing were cut with a cryostat blade while frozen into 1-mm frozen slabs. While frozen, the region of the nucleus caudatus and septum surrounding the SVZ was dissected and immediately lysed with a Micro-homogenizer (EMS, Hatfield, PA) in protein extraction buffer, 10-mM Tris-HCl, 1% sodium dodecyl sulfate (SDS) buffer solution (pH 7.4) on ice, then quantitated with a BCA Micro-Protein Quantification Kit (Pierce, Waltham, MA). Immediately before loading, 30-µg samples of brain extract were diluted in loading buffer (4% SDS, 20% glycerol, 0.02% bromphenol blue, 200-mM dithiothreitol in 0.5 M Tris-HCl, pH 6.8), heated at 95°C for 5 minutes and cooled on ice for 5 minutes.
To visualize COX-2 enzyme, proteins were separated on a 4%–20% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA), electroblotted onto polyvinyl difluoride membranes (Bio-Rad, Hercules, CA), and blocked overnight with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) in Tris-buffered saline/Tween 20 buffer (Sigma-Aldrich). To detect COX-2, a rabbit polyclonal primary antibody (Novus) was used at a dilution of 1:1000. To detect β-actin, a mouse monoclonal primary antibody (Novus biological, Littleton, CO) at a dilution of 1:5000 was used. Secondary antibodies were either HRP-coupled goat anti-rabbit IgG (for COX-2) or HRP-coupled goat anti-mouse (for β-actin) (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA), both diluted at 1:10,000. The blots were developed using the enhanced ECL reaction (Pierce) and processed by x-ray. The density of bands at the appropriate molecular weights for these proteins on each gel was normalized for β-actin across gels to bands and compared by Image J. The results were transformed to percentage of actin band intensity.
Rat recombinant IL-6 (Shenandoah Biotechnology, Warwick, PA) was prepared by dissolving 10 μg in 100 μL of 10-mM HCl, and then neutralizing with phosphate-buffered saline to pH 7.4, giving final concentrations of 1, 1.5, and 2.5 μg, each in 50 μL, for each of 3 planned injections. Vehicle solution for control injections was prepared in the same manner, omitting the IL-6.
Data are presented as mean ± standard error of the mean or median (interquartile range [IQR]). Analysis of variance for repeated measures was used to analyze temperature differences. The model included a time-group interaction term. Time zero measurements were not included in the analysis and no pairwise comparisons across individual time points were performed.
Maternal IL-6 serum levels were normally distributed (Kolmogorov–Smirnov test, P = .662) and were analyzed by Student t test. Number of pixels occupied by ED-1/CD68 immunoreactive cells and COX-2 immunoreactive densities in the SVZ were all compared by Mann-Whitney U test between saline-injected and IL-injected animals. For COX-2 Western blotting, data were compared with Fisher exact test. Sample sizes were based on preliminary experiments (data not shown) that suggested >90% power to detect a difference in peak temperature between groups of 0.4°C with n = 5 pups per group. To increase the number of fetal brains available for analysis and to allow for tissue loss, 12 dams per group were studied. A sample size of 6 fetal brains/group also provided >90% power to detect a difference of 30 ED-1–stained cells. Statistical analyses were performed with SAS version 9.4 or JMP Pro version 11 (both from the SAS Institute, Cary, NC). A P value of <.05 was considered statistically significant.
All animals tolerated the injection procedures and no premature delivery occurred. Additional animals allowed to proceed to term delivered live pups that did not differ between IL-6 and vehicle groups in birth weight (data not shown). Injection of IL-6 into near-term pregnant dams induced maternal temperature elevation compared to vehicle injection (Figure 1). The mean difference in temperature between IL-6 and vehicle groups (mean [95% confidence interval]) was 0.516°C (0.1–0.931), P = .0197. Overall, the effect of IL-6 was statistically significant (repeated measures analysis of variance, P < .0001).
IL-6 serum levels were significantly higher in drug-injected compared to vehicle-injected animals (mean ± standard deviation: 923 ± 97 vs 143 ± 94 pg/mL, P = .0006). Neonatal microglial activation, as shown by ED-1–stained cells, in the caudal hippocampus (Figure 2A, B) and septal areas (Figure 2C, D) was significantly higher in offspring whose mothers received IL injections compared to saline-exposed animals (median [IQR]: caudal hippocampus, 99 [94–104] and 64 [57–68], respectively, P = .002; lateral septum, 102 [96–111] and 68 [65–69], respectively, P = .002) (Figure 2B, D; Table).
Likewise, brain areas were significantly more inflamed in offspring of dams that received IL-6 compared to animals exposed to saline, as shown by COX-2 immunostaining in the dorsal hippocampus (median [IQR] pixel intensity/mm2: IL-6 exposed 27 [22–32] versus saline exposed 16 [14–19], respectively, P = .013) and the lateral septum (IL-6 exposed 22 [20–26] and saline exposed 16 [14–19], respectively, P = .05; Table). Quantitative COX-2 Western blotting activity in the nucleus caudatus was significantly increased by IL-6 treatment (vehicle, 0% of β-actin intensity versus IL-6, mean ± standard error of the mean, 41.5% ± 24%, Fisher exact test, P < .001; Figure 3).
In Saline brains, COX2 immunoreactivity was near the threshold of detection (Figure 2E), whereas in IL-6 brains the COX2 signal was intense, localized primarily in the lateral septum and dorsal hippocampus, and in vascular profiles throughout the brain parenchyma immediately deep to the SVZ (Figure 2F).
The results of these investigations show that it is possible to induce noninfectious temperature increase in near-term pregnant rats with an inflammatory stimulus, injection of systemic IL-6. The pups of treated rats develop signs of significant neuroinflammation. The treatment does not result in gross behavioral abnormalities in the dams and does not induce labor. In these respects, we believe that this preparation may be a partial model of the phenomenon of labor epidural–associated maternal fever and its possible consequences for the fetus and newborn.
Labor epidural analgesia induces fever in approximately one-quarter of women,1 and the etiology appears to be noninfectious inflammation. A number of lines of evidence support this etiology. First, increasing duration of exposure to labor epidural analgesia is associated with increasing levels of maternal IL-6.10,20 Second, placental inflammation is a prominent feature in most cases of epidural-associated fever.21 Acetaminophen, which is antipyretic but not strongly anti-inflammatory, fails to block epidural-associated fever,14 but maternal corticosteroids do block it.13 The noninfectious etiology is also supported by the failure of broad-spectrum antibiotics to prevent fever with epidural analgesia.15 Furthermore, meticulous efforts to identify pathogenic bacteria in the placenta by culture or the polymerase chain reaction have failed to identify such organisms in febrile women with epidural analgesia and placental inflammation.12,22 The model described in the present investigation mirrors these observations, as it induces increase in temperature through a noninfectious inflammatory stimulus at physiologically relevant concentrations of IL-6.
The neonatal effects of maternal intrapartum fever are potentially severe. In the immediate postpartum period, lower Apgar scores, neonatal tone,23 increased need for bag-mask ventilation,24 chest compressions23,24 and intubation,25 and increased otherwise unexplained neonatal seizures5 have been associated with maternal fever or even subfebrile temperature elevation.26 In addition, maternal intrapartum fever and chorioamnionitis are significant risk factors for subsequent development of cerebral palsy.27–30
There are no complete animal models of labor epidural analgesia, but rodent and rabbit models of infectious maternal intrapartum inflammation clearly demonstrate neuroinflammation and cellular damage in the fetus.9,31 Similar lesions are seen when infectious mediators such as lipopolysaccharide are injected into the mother.32,33 The anti-inflammatory cytokine IL-10 can block these effects by reducing microglial activation, again pointing to inflammatory-mediated injury.34,35 Because epidural-associated fever is noninfectious, we modeled inflammatory intrapartum fever with IL-6 injected shortly before term gestation in the pregnant rat. The level of IL-6 achieved in the treated dams approximates those seen in laboring humans with epidural analgesia.10 However, it should be noted that rat neurodevelopment is not entirely synchronous with human neurodevelopment, and term in rats is roughly equivalent to late preterm in humans.36
The results of these investigations show that this stimulus can induce neonatal brain inflammation. The brain regions studied showed inflammation in the periventricular SVZ, where precursors of neurons, astrocytes, and oligodendroglia arise from tripotent neural stem cells located in the lateral and dorsal wall of the lateral ventricle. The mechanism of this injury was not directly investigated in this preliminary study, but it is likely due to induction of fetal inflammation, rather than direct transplacental passage of maternal IL-6. In midgestation rats, IL-6 freely crosses the placenta, but such passage is minimal at term.37,38 Conversely, maternal infection or other inflammation appears to induce fetal inflammation distant from the original source of infection.39 Activation of microglia, as seen in our model, is thought to play a pivotal early role in brain injury, including in human brain.34,39 Moreover, activation of COX-2, as seen in our model, is thought to play an important role in newborn neuroinflammatory brain injury.40,41
Other investigations have modeled the effect of excess maternal or fetal IL-6 on the developing fetal brain. Smith et al42 demonstrated induction of fetal neuroinflammation and behavioral abnormalities in offspring of mice injected at midgestation with very high doses of IL-6. Similarly, Samuelsson et al43 showed that injection of IL-6 over multiple days of mid- or late-gestation rats led to increased neuroinflammation in the offspring, increased IL-6 production in the neonatal brain, and impaired learning behaviors. Heyser et al44 demonstrated impaired avoidance learning in transgenic mice that overexpressed IL-6 in the brain. Our model complements these efforts by more closely mirroring the clinical situation of epidural-associated fever, in which a brief moderate inflammatory stimulus is seen in the mother in the peripartum period.
There are several limitations to our model. Most important, while the phenomenon of epidural-associated fever is well characterized, the link between epidural analgesia and elevated IL-6 is not understood. Thus our model focuses on the potential consequences of maternal noninfectious inflammation, but does not address how such inflammation develops. Furthermore, while we have demonstrated neuroinflammation from physiologically relevant levels of maternal IL-6 exposure, we have not yet measured functional or anatomical consequences in the newborn. This is an area of active investigation by our group. Third, we cannot be certain that maternally administered IL-6 is not reaching the fetus directly by transplacental passage, though as noted above, other work suggests such transport is minimal at term. Fourth, we did not evaluate the effect of maternal IL-6 on the placenta. Placental inflammation is a consistent feature of epidural-associated fever in humans, though it is unclear whether this is a source or a result of systemic inflammation. This is another area of active investigation by our group. Finally, we did not consider sex differences in fetal effects of maternal IL-6 exposure, and the investigation was underpowered to detect such differences should they have been present. There is evidence of enhanced sensitivity to inflammatory damage in males including in human disease.45 However, we believe that inclusion of fetuses from both sexes would have biased our results toward showing a smaller response rather than an exaggerated one, and the overall results were nonetheless statistically significant.
In summary, we have developed a rat model to study the consequences of maternal noninfectious inflammation at term on the fetal brain. Potentially, this model could be used to study functional consequences of such inflammation in the newborn as well as providing a platform for studying putative strategies for preventing or modulating epidural-associated inflammation and fever.
Name: Scott Segal, MD, MHCM.
Contribution: This author helped to conceive the study, design the study, and helped with statistical oversight, and preparation and review of the manuscript.
Name: Carlo Pancaro, MD.
Contribution: This author helped to design and execute the experiment, and prepare and review the manuscript.
Name: Iwona Bonney, PhD.
Contribution: This author helped to execute the experiment and review the manuscript.
Name: James E. Marchand, PhD.
Contribution: This author helped to design and execute the experiment, helped with compliance and animal welfare, provided technical oversight, helped prepare the manuscript, and helped review the manuscript.
This manuscript was handled by: Alexander Zarbock, MD.
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