Objective
We hypothesized that repetitive umbilical cord occlusions (UCOs) with worsening acidosis will lead to a fetal inflammatory response.
Study Design
Chronically instrumented fetal sheep underwent a series of UCOs until fetal arterial pH decreased to <7.00. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1B and IL-6 levels. Animals were euthanized at 24 hours of recovery with brain tissue processed for subsequent measurement of microglia and mast cell counts.
Results
Repetitive UCOs resulted in a severe degree of fetal acidemia. Fetal plasma IL-1B values were increased ∼2-fold when measured at maximal fetal acidosis and again at 1-2 hours of recovery. Fetal microglia cells were increased ∼2-fold in the white matter and hippocampus, while mast cells were increased ∼2-fold in the choroid plexus and now evident in the thalamus when analyzed at 24 hours recovery.
Conclusion
Repetitive UCOs leading to severe acidemia in the ovine fetus near term will result in an inflammatory response both systemically and locally within the brain.
Birth asphyxia with severe fetal acidemia, defined as an umbilical artery pH <7.00, is associated with increased risk for newborn hypoxic-ischemic encephalopathy (HIE), although the majority of these infants will still be without noted complications. This indicates that birth asphyxia with resultant brain injury in most instances is multifactorial in basis with gestational age at birth, duration of hypoxic acidemia, fetal/newborn compensatory capacity, and newborn resuscitation also likely to be contributory.
There is now considerable epidemiologic and clinical evidence that increases in inflammatory cytokines during the course of infection play a contributing role in the increased risk for brain injury, whether intrauterine with chorioamnionitis preterm or at term, or postnatal in the neonate. This has resulted in a number of animal-based studies with the induction of perinatal infection and/or inflammatory response by bacterial products further implicating a contributory role for an increase in fetal inflammatory cytokines along with an increase in inflammatory cells within the brain, in resultant brain injury. These animal-based studies furthermore show an interactive effect whereby bacterial endotoxin sensitizes the immature brain to hypoxic-ischemic injury indicating that infection and hypoxic acidemia may have a synergistic role in causing fetal brain injury. There is also considerable clinical and experimental evidence that increases in inflammatory mediators play a contributory role in the pathogenesis of newborn HIE in the absence of overt infection, although most cases of histopathologic chorioamnionitis will be subclinical especially at term. In addition, hypoxia and hypoperfusion both lead to increases in cytokine expression and/or production within the placenta supporting the contention that reduced uterine or umbilical blood flow with contractions through labor might lead to an increase in inflammatory cytokines as well as worsening fetal acidosis.
Variable-type fetal heart rate (FHR) decelerations due to umbilical cord compression with acute reduction in fetal oxygenation are the most common nonreassuring FHR pattern observed intrapartum. Although these short-term hypoxic episodes are generally well tolerated, when more frequent and/or severe they have been associated with an increased incidence of neonatal acidosis, low Apgar scores, and nuchal cord involvement at the time of delivery. We have therefore used the chronically catheterized ovine fetus near term to test the hypothesis that repetitive cord occlusions with worsening acidosis as might be seen clinically during labor will lead to an inflammatory response both systemically and locally within the brain. The proinflammatory cytokines interleukin (IL)-1B and IL-6 have been determined as measures of systemic inflammation because these cytokines play a prominent regulatory role in the inflammatory response and have been shown to increase as part of the fetal/neonatal inflammatory response to infection and with HIE. The distribution of microglia and mast cells within the brain has been determined as a measure of local inflammation because these cells also play a prominent role in the inflammatory response and likewise have been shown to increase with fetal/neonatal infection and/or hypoxia.
Materials and Methods
Surgical preparation
Ten near-term (125 ± 1 days’ gestation) fetal sheep of mixed breed were surgically instrumented (term = 145 days). The anesthetic and surgical procedures and postoperative care of the animals have previously been described. Briefly, using sterile technique under general anesthesia, the upper body of the fetus and proximal portion of the umbilical cord were exteriorized through an incision in the uterine wall. Polyvinyl catheters (Bolab, Lake Havasu City, AZ) were placed in the right and left brachiocephalic arteries, and the right brachiocephalic vein. Stainless steel electrodes were implanted biparietally on the dura for the recording of electrocortical activity and over the sternum for recording electrocardiographic (ECG) activity. An inflatable silicone occluder cuff (OCHD16; In Vivo Metric, Healdsburg, CA) was positioned around the proximal portion of the umbilical cord and secured to the abdominal skin. Once the fetus was returned to the uterus, a catheter was placed in the amniotic fluid cavity and subsequently in the maternal femoral vein.
Animals were allowed a 3- to 4-day postoperative period before experimentation, during which antibiotics were given and catheters were flushed with heparinized saline to maintain patency. Animal care followed the guidelines of the Canadian Council on Animal Care and was approved by the University of Western Ontario Council on Animal Care.
Experimental protocol
Animals were studied through a 1- to 2-hour control period and an experimental period of repetitive umbilical cord occlusions (UCOs) with worsening acidemia, and were then allowed to recover overnight ( Figure 1 ). A computerized data acquisition system was used to record pressures in the fetal brachiocephalic artery and amniotic cavity, and the electrical signals for electrocortical and ECG activities, which were monitored continuously through the control and experimental periods, and first 2 hours of the recovery period (Chart 5 for Windows; AD Instruments Pty Ltd, Castle Hill, Australia).
After the baseline control period that began at ∼0800 hours, repetitive UCOs were performed with increasing severity until severe fetal acidemia was detected (arterial pH <7.00), at which time the UCOs were stopped. UCO was induced by complete inflation of the occluder cuff with ∼5 mL of saline solution that was previously determined by visual inspection and testing at the time of surgery. During the first hour a mild UCO series was performed consisting of cord occlusion for 1-minute duration every 5 minutes. During the second hour a moderate UCO series was performed consisting of cord occlusion for 1-minute duration every 3 minutes. During the third hour a severe UCO series was performed consisting of cord occlusion for 1-minute duration every 2 minutes and this series was continued until the targeted fetal arterial pH was attained. Following the mild and moderate UCO series a 5- to 10-minute period with no UCO was undertaken, during which fetal arterial blood was sampled and arterial blood pressure, electrocortical, and ECG data were recorded in the absence of FHR decelerations. After attaining the targeted fetal arterial pH <7.00 and stopping the repetitive UCOs, animals were allowed to recover for ∼24 hours.
Fetal arterial blood samples were obtained during the baseline control period (3 mL), at the end of the first UCO of each UCO series (1 mL), and ∼5 minutes after each UCO series (3 mL). In addition, fetal arterial blood samples were obtained between UCOs at ∼20 and 40 minutes of the moderate and severe UCO series (1 mL), and at 1, 2, and 24 hours of recovery (3 mL). Maternal venous blood samples were also obtained during the baseline control period, and at 1 and 24 hours of recovery (3 mL). All fetal blood samples were analyzed for blood gas values, pH, glucose, and lactate with an ABL-725 blood gas analyzer (Radiometer Medical, Copenhagen, Denmark) with temperature corrected to 39.0°C. Fetal and maternal 3-mL blood samples at selected time points ( Table ) were spun at 4°C (4 minutes, 4000 g force; Beckman TJ-6; Beckman Coulter, Inc, Fullerton, CA) and the plasma decanted and stored at –80°C for subsequent cytokine analysis.
| Variable | Baseline n = 10 | Maximal fetal acidosis n = 10 | Early recovery 1-2 h n = 9 | Late recovery 24 h n = 9 |
|---|---|---|---|---|
| Fetal | ||||
| IL-1B | 525 ± 96 | 1068 ± 167 a | 967 ± 152 a | 640 ± 143 |
| IL-6 | 429 ± 50 | 460 ± 78 | 446 ± 58 | 440 ± 102 |
| Maternal | ||||
| IL-1B | 379 ± 64 | 294 ± 64 | ||
| IL-6 | 563 ± 160 | 682 ± 243 |
After the 24-hour recovery blood sample, the ewe and the fetus were killed by an overdose of barbiturate (30 mg of sodium pentobarbital intravenously; MTC Pharmaceuticals, Cambridge, Ontario, Canada) and a postmortem examination was carried out during which time fetal sex and weight were determined, and the location and function of the umbilical cord occluder cuff were confirmed. The fetal brain was then perfusion fixed with 500 mL of cold saline followed by 500 mL of 4% paraformaldehyde and processed for histochemical analysis as we have previously reported. To obtain brain tissue from control animals for comparative purposes to that of the repetitive UCO animals, 2 noninstrumented twins of instrumented experimental group animals and 4 instrumented control animals from a separate study were used. These latter animals were similarly instrumented and of the same gestational age as the repetitive UCO animals and all animals underwent the same perfusion-fixation procedure and brain tissue processing for histochemical study.
Plasma cytokine and tissue histochemical analysis
An enzyme-linked immunosorbent assay was used to analyze in duplicate the concentrations of IL-1B and IL-6 in fetal arterial and maternal venous plasma samples. IL-1B and IL-6 standards were purchased from the University of Melbourne, Center of Animal Biotechnology (Melbourne, Australia). Mouse antiovine IL-1B (MAB 1001) and IL-6 (MAB 1004) monoclonal antibodies and rabbit antiovine IL-1B (AB 1838) and IL-6 (AB 1889) polyclonal antibodies were purchased from Chemicon International (Temecula, CA). Separate 96-well plates were coated with mouse monoclonal ovine IL-1B or IL-6 antibody (1:200, in 0.1 mol/L of NaCO 3 , pH to 9.6) and incubated overnight at 4°C. The following day, plates were washed 3 times with wash solution (phosphate-buffered saline [PBS] with 0.05% Tween, pH to 7.4) to remove excess monoclonal antibody. Plates were then blocked with assay diluent (555213, BD OptEIA; BD Biosciences, San Jose, CA) at room temperature for 1 hour. Wells were then rinsed 3 times with the wash solution followed by aliquoting standards (40,000-156 pg/mL and blanks) and samples, and incubation on the shaker at room temperature for 2 hours. Subsequently, wells were rinsed 3 times with washing solution and the appropriate rabbit antiovine polyclonal antibody (IL-1B or IL-6, 1:500) was added to each well and incubated on the shaker for 1 hour. Following ≥5 washes, HRP-donkey antirabbit IgG (AP182p, 1:10,000; Chemicon International) was added to each well and incubated on the shaker for 1 hour. The wells were then washed 7 times with wash solution to remove all unbound secondary antibody, followed by the addition and 30-minute incubation with substrate solution (51-2606KC and 51-2607KC; BD Biosciences, Mississauga, Ontario, Canada) in the dark. Stop solution (1N H 2 SO 4 ) was applied and each well was read using a spectrophotometer at 450 nm, with 575-nm wavelength correction.
The presence of microglia in brain tissue was determined by avidin-biotin-peroxidase complex enhanced immunohistochemistry (Vectastain Elite; Vector Laboratories Inc, Burlingame, CA). To reduce staining variability, all immunohistochemistry was performed on the same day with the same batch of antibody and solutions. Tissue sections were deparaffinized in 3 sequential xylene baths for 5 minutes each and subsequently rehydrated in a series of alcohol baths (100%, 90%, 70%) each lasting 2 minutes followed by a 5-minute rinse in tap water and equilibration in PBS. Antigen retrieval was performed by incubation of the sections in boiling citrate buffer (2.1 g of citric acid/1 L of water; pH 6.0 with concentrated NaOH) in a steamer for 30 minutes followed by a 35-minute cooling period in the citrate buffer outside of the steamer and a 5-minute rinse in tap water. Sections were then washed 3 times in PBS before and after endogenous peroxidases were quenched by a 10-minute bath in 3% hydrogen peroxide in PBS. Avidin-biotin blocking was performed with a 15-minute incubation first with avidin followed by biotin. Nonspecific protein binding was blocked with a 10-minute incubation in background sniper blocking serum (Biocare Medical, Concord, CA). Sections were then incubated with an anti-ionized calcium-binding adaptor molecule (IBA)1 rabbit polyclonal antibody (1:500; Wako Industries, Richmond, VA) diluted in Dako diluent solution (Dako Cytomation, Carpinteria, CA) overnight at 4°C, which has been reported to be a robust marker for microglia in human and animal studies. Sections were subsequently rinsed 3 times for 5 minutes in PBS and then incubated with secondary antibody (1:200, biotinylated antirabbit immunoglobulin G; Vector Laboratories Inc) at room temperature for 30 minutes and rinsed as described earlier. Sections were then incubated with streptavidin/biotin/peroxidase/reagent (Vectastain ABC Elite; Vector Laboratories Inc) at room temperature for 45 minutes. The detection of bound antibody was obtained with a 2-minute incubation in Cardassian DAB Chromogen (Biocare Medical) at room temperature. Sections were rinsed with running tap water for 5 minutes, then dehydrated in 5 brief alcohol baths of increasing concentration (1 bath in 70% alcohol, 2 baths in 90%, and 2 baths in absolute alcohol), followed by 3 xylene baths of 5 minutes each before being cover slipped in Permount (Fisher Scientific, Ottawa, Ontario, Canada). To demonstrate nonspecific binding, additional negative control sections were processed as described, with the exception that the primary antibody was omitted.
The presence of mast cells in brain tissue was determined using histologic and morphologic assessment techniques. Tissue sections were deparaffinized in 3 sequential xylene baths for 5 minutes each and subsequently rehydrated in a series of alcohol baths (100%, 90%, 70%) each lasting 2 minutes followed by a 5-minute rinse in tap water and equilibration in PBS. Sections were then stained in 0.1 mol/L of hydrochloric acid with toluidine blue (pH = 2) for 10 minutes and then rinsed with running tap water for 5 minutes. Sections were quickly rinsed in acetic acid, then dehydrated in 5 brief alcohol baths of increasing concentration (1 bath in 70% alcohol, 2 baths in 90%, and 2 baths in absolute alcohol), followed by 3 xylene baths of 5 minutes each before being cover slipped in Permount (Fisher Scientific).
Brain regions that were selected from each animal for analysis were taken from a coronal section of blocked cerebral hemisphere tissue at the level of the mamillary bodies and included the parasagittal and convexity cerebral gray matter and leptomeninges, periventricular white matter, thalamus, choroid plexus, and the combined CA2 and CA3 regions of the hippocampus. Each of the gray matter regions was further divided into subregions combining layers 1, 2, and 3 and layers 4, 5, and 6. After showing no significant difference between these subregions, all layers were combined to represent the gray matter. Image analysis was performed with a transmitted light microscope (Leica DMRB; Leica-Microsystems, Wetzler, Germany) at ×40 magnification. Positive microglia cell immunostaining was quantified with an image analysis program (Image Pro Plus 6.0; Media Cybernetics, Silver Spring, MD). The image analysis system was first calibrated for the magnification settings that were used, and thresholds were established to provide even lighting and no background signal. Six high-power field (HPF) photomicrographs (HPF area = 7 cm 2 ) per brain region/subregion per animal were collected as a 24-bit RGB color modeled image. The same illumination setting was applied to all images for all of the brain regions, therefore allowing for comparison within each brain region (ie, control vs repetitive cord occlusion animal groups), and between brain regions (ie, gray matter vs white matter). For the microglia analysis, and using the Image Pro Plus RGB color range selection tool (Media Cybernetics), color sampling of positive DAB-stained areas was obtained from multiple brain regions of control and UCO animals, and tested for specificity against the negative control. Appropriate ranges of color were selected showing positive contiguous cytoplasmic staining as criteria for microglia cell count scoring, which were then applied uniformly to calibrated images for all brain regions ( Figure 2 ). Scoring was performed in a blinded fashion to experimental groups. For mast cell analysis, scoring was performed manually based on positive stain and characteristic morphology with the presence of large metachromatic secretory granules filling the cytoplasm due to the presence of sulfonated proteoglycans such as heparin, and a unilobular ovoid nucleus ( Figure 2 ). Scoring was again performed in a blinded fashion to experimental groups.
