Objective
Maternal infection or inflammation may induce fetal inflammatory responses associated with fetal injury and cerebral palsy. We sought to assess the inflammation-associated neuroprotective potential of prophylactic N-acetyl-cysteine (NAC). We examined the effect of NAC on prevention of maternal lipopolysaccharide (LPS)-induced neonatal brain injury using magnetic resonance imaging.
Study Design
Pregnant Sprague Dawley dams (n = 5-8) at embryonic day 18 received intraperitoneal injection of LPS or saline at time 0. Animals were randomized to receive 2 intravenous injections of NAC or saline (time −30 and 120 minutes). Pups were delivered spontaneously and allowed to mature until postnatal day 25. Female offspring were examined by magnetic resonance brain imaging and analyzed using voxel-based analysis after spatial normalization. T2 relaxation time was used to assess white matter injury and diffusion tensor imaging for apparent diffusion coefficient (ADC) to assess white and gray matter injury.
Results
Offspring of LPS-treated dams exhibited significantly increased T2 levels and increased ADC levels in white and gray matter (eg, hypothalamus, motor cortex, corpus callosum, thalamus, hippocampus), consistent with diffuse cerebral injury. In contrast, offspring of NAC-treated LPS dams demonstrated similar T2 and ADC levels as control in both white and gray matter.
Conclusion
Maternal NAC treatment significantly reduced evidence of neonatal brain injury associated with maternal LPS. These studies suggest that maternal NAC therapy may be effective in human deliveries associated with maternal/fetal inflammation.
Cerebral palsy (CP) defines a group of nonprogressive motor impairment syndromes that manifest during fetal or early postnatal life. With an incidence of 2.5 per 1000 births, the rate of CP has remained unchanged over the past decades despite marked improvements in obstetrical and neonatal care. Whereas CP was previously attributed to intrapartum hypoxia, asphyxia actually accounts for only a small percentage of CP cases. Recent studies have convincingly demonstrated the association of fetal/amniotic infection with CP as well as other neurodevelopmental disorders such as schizophrenia and Parkinson’s disease. However, the etiology for the majority of CP cases remains unknown.
Most recently, epidemiological studies suggest that maternal infections, including urinary tract and periodontal infections, may be associated with intrauterine inflammation, preterm birth, low birthweight, preeclampsia, and cerebral palsy. The mechanisms by which maternal or fetal infections jeopardize fetal development are unclear, although bacterial lipopolysaccharide (LPS), reactive oxygen species, and proinflammatory cytokines have been implicated in the pathogenesis.
LPS can directly induce proinflammatory (eg, interleukin [IL]-6) and antiinflammatory (eg, IL-10) cytokines. LPS further induces mediators of oxidative stress that themselves may induce cytokine production. Oxidative stress leads to cytokine induction by activating nuclear factor-kappa B (NFκB), which is a major transcription factor for the proinflammatory cytokines. The excessive production and release of cytokines in the fetus have been linked to fetal brain injury. In vivo intracerebral injection of tumor necrosis factor (TNF)-α or IL-1β into the white matter of newborn rats induced microglial activation, hemorrhage, and myelin damage.
Findings from in vitro experiments indicate that proinflammatory cytokines may have a direct effect on oligodendrocytes; TNF-α induces apoptotic death of oligodendrocytes in culture, and blocking antibodies against TNF-α or IL-1β protect oligodendrocytes against bacterial endotoxin-induced cell death in explants of immature optic nerves.
Enhancing the activity or the availability of antioxidants may modulate the inflammatory response during pregnancy, thereby reducing oxidative stress and the risk to the developing fetal brain injury. We previously demonstrated that maternal LPS can induce an acute increase in the proinflammatory cytokines in the amniotic fluid, fetal blood, and the fetal brain. We further demonstrated that N-acetyl-cysteine (NAC), an antioxidant shown to inhibit NFκB activity in culture, decreased the acute inflammatory response.
In the current study, we have sought to determine the long-term effect of maternal inflammation on the development of white and gray matter injury in the offspring brain and to assess the protective effects of NAC on maternal LPS-induced neonatal brain injury using diffusion tensor imaging (DTI). This technique is based on the measurements of water diffusion in the tissue for demonstrating white matter brain injury. It appears to be superior to standard magnetic resonance techniques for demonstrating white matter brain injury. We hypothesized that maternal NAC will attenuate the long-term offspring brain injury associated with maternal inflammation.
Materials and Methods
Animals and treatments
Sprague-Dawley pregnant rats (Harlan Sprague Dawley, Inc, Chicago, IL) were obtained at gestational day 15 and 17 (term = 21 days) and allowed to acclimate for 72 hours prior to the beginning of experiments. Animals were maintained in temperature- (37°C) and light (6:00 am lights on; 6:00 pm lights off)-controlled facilities with access to food (LabDiet 5001 Rodent Diet; PMI Nutrition International, LLC, Brentwood, MO) and water ad libitum throughout the study. NAC (Sigma, St. Louis, MO) was reconstituted in physiological saline at a pH of 6.8-7.2 and administered intravenously (IV) at 300 mg/kg body weight (bw), LPS ( Escherichia coli, serotype 0111:B5; Calbiochem, La Jolla, CA) was reconstituted in physiological saline and administered intraperitoneally at 500 μg/kg bw. At gestational day 18, the pregnant rats received injection of LPS (500 μg/kg, intraperitoneally) or saline intraperitoneally, at time 0.
The animals were randomized to receive 2 IV injections (tail vein) of NAC (300 mg/kg) (LPS-NAC: NAC-LPS-NAC; n = 5) or saline (LPS: Sal-LPS-Sa; n = 8) at time −30 minutes and at time 150 minutes. An additional group of animals received 3 doses of saline (control; n = 6).
The protocols and procedures for this study were approved by the Institutional Animal Care and Utilization Committee at Rappaport Research and Education Institute. Pups were delivered spontaneously (embryonic day 21) and allowed to mature until postnatal day 25. In the present study, we examined only young female rats before sexual maturity. Female offspring at postnatal day 25, 1 from each dam (5-8 per group), were examined by brain magnetic resonance imaging (MRI).
Magnetic resonance imaging protocol
MRI scan of rats’ brains were performed on a 7T Bruker MRI machine (Bruker, Billerica, MA). The rats were anesthetized with 1-2% isoflurane and oxygen and maintained in 37°C, and their breathing was monitored with a breathing sensor. The DTI and T2 relaxation protocols were performed.
DTI parameters
These parameters included the following: spin echo sequence, repetition time (TR) = 4000 milliseconds; echo time (TE) = 25 milliseconds; slice thickness = 1 millimeter; δ = 4.5; Δ = 10, 15 gradient directions; B value = 1000 meters; matrix: 128 × 96.
T2 parameters
The T2 parameters included the following: MSME sequence, TR = 3000 milliseconds, 16 different TE (milliseconds):10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160; field of view = 1.920 cm; matrix: 256 × 128; slice thickness = 1 millimeter.
Image analyses
DTI image analysis
The apparent diffusion coefficient (ADC) maps and T2 maps were calculated for each rat from the magnetic resonance images. All maps were coregistered and normalized to a template (based on 1 subject) and smoothed (2 mm FWHM) using the SPM software version 2 (SPM Technologies Inc, Morganville, NJ).
T2 relaxation image analysis
The T2 map was extracted for each rat from the T2 relaxation maps.
Statistical analyses
Voxel-based analysis (VBA) is a whole-brain statistical technique that allows the detection of regionally specific differences in brain tissue composition on a voxel-by-voxel basis. VBA involves transforming the images into a standard space (spatial normalization), using an automated nonrigid registration of the images to an anatomical template. After normalization, an analysis of variance was performed voxel by voxel to compare the groups and to assess white matter injury or any brain tissue changes. Only significant regions that passed the threshold of P < .001 are presented.
Region of interest (ROI) analysis
The ROI analysis was performed to extract the parameters within regions showing differences between ADC/T2 of LPS vs LPS-NAC rats.
In the graphs and tables, the parameters extracted are the average ADC/T2 comparing the LPS, LPS-NAC, Control, and Control-NAC groups.
Results
Number of newborn pups
There was a trend toward fewer newborn pups in the LPS group compared with the LPS-NAC group (8 ± 5 vs 12 ± 1 pups, P = .08). No difference was demonstrated between the LPS-NAC group and the Control group (11 ± 2).
LPS offspring
Differences in neonate T2 brain maps
Maternal LPS injection (intraperitoneally [ip]) increased T2 levels (less dense tissue) in white and gray matter areas in offspring . T2 was significantly higher in the LPS group compared with the Control group in the following regions: visual cortex, cingulate cortex, periaqueductal gray (PAG), dorsal hippocampal commissure (DHC), corpus callosum (CC), external capsule (EC), dentate gyrus (DG), substantia nigra (SN) geniculate body (GenB), hippocampus (Hip), auditory cortex (AudC), piriform cortex (piriC), cingulum (cingo), thalamus, reticular thalamic nucleus (Th-rt), caudate putmen (CPu), insular cortex (insular cor), and the CA3 ( P < .001). Conversely, no area was found with increased T2 levels in the Control group compared with the LPS group.
Differences in ADC maps
We found that ADC was significantly higher in the LPS group compared with the Control group in the following regions: posterior thalamic nucleus (Th PO), ventroposterior-medial nucleus of the thalamus (Th VPM), hypothalamus, motor cortex (M1/M2), similar to the T2 results. No area was found with higher ADC levels in the control group compared with the LPS group.
LPS-NAC offspring
Offspring of NAC-treated LPS dams demonstrated decreased T2 levels (more dense tissue) as compared with the offspring of the LPS dams in all areas of the brain affected by LPS (visual cortex, cingulate cortex, PAG, DHC, CC, EC, DG, SN, GenB, Hip, AudC, piriC, cingo, thalamus, Th-rt, CPu, insular cor, and the CA3) and decreased ADC in all areas of the brain affected by LPS (Th PO, Th VPM, hypothalamus, and M1/M2. Offspring of LPS-NAC dams demonstrated similar T2 and ADC levels as the Control in both white and gray matter ( Figures 1 and 2 ).
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