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Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats
Published on: November 20, 2015
Caspase activation in fetal rat brain following experimental intrauterine inflammation
Aditi Sharangpani1, Asako Takanohashi, Michael J Bell
1Division of Critical Care Medicine and Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, George Washington University School of Medicine, Washington, DC 20010, USA.
Insights
Intrauterine inflammation causes fetal brain injury and cell death via apoptosis. This study shows both extrinsic and intrinsic pathways, involving Fas, are activated, offering insights for preventing developmental brain injuries like cerebral palsy.
Area of Science:
- Neuroscience
- Developmental Biology
- Immunology
Background:
- Intrauterine inflammation is linked to fetal brain injuries, including periventricular leukomalacia (PVL) and cerebral palsy (CP).
- Previous research in a rat model showed lipopolysaccharide (LPS)-induced inflammation leads to fetal brain apoptosis and dysmyelination.
Purpose of the Study:
- To investigate the specific cell death pathways involved in intrauterine inflammation-induced fetal brain injury.
- To determine if the extrinsic apoptosis pathway, involving Fas receptor activation, contributes to cell death in this model.
Main Methods:
- Pregnant rats were administered LPS intracervically at embryonic day 15.
- Immunoblotting, immunohistochemistry, and immunoprecipitation were used to analyze caspase activation and the death-inducing signaling complex (DISC).
Main Results:
- Activated caspase-3 was detected 24 hours after LPS exposure.
- Caspase activity assays revealed rapid increases in caspases-9, -10, -8, and -3 following LPS administration.
- Activated caspase-3 and Fas-positive cells were found in the developing white matter 24 hours post-LPS, and the DISC complex was detected within 30 minutes.
Conclusions:
- Apoptosis in this intrauterine inflammation model involves both extrinsic and intrinsic pathways.
- Fas receptor activation appears to play a role in the observed cell death.
- Understanding these mechanisms can inform strategies to mitigate fetal brain injury in children.
Abstract:
Intrauterine inflammation has been implicated in developmental brain injuries, including the development of periventricular leukomalacia (PVL) and cerebral palsy (CP). Previous studies in our rat model of intrauterine inflammation demonstrated apoptotic cell death in fetal brains within the first 5 days after lipopolysaccharide (LPS) administration to mothers and eventual dysmyelination. Cysteine-containing, aspartate-specific proteases, or caspases, are proteins involved with apoptosis through both intracellular (intrinsic pathway) and extracellular (extrinsic pathway) mechanisms. We hypothesized that cell death in our model would occur mainly via activation of the extrinsic pathway. We further hypothesized that Fas, a member of the tumor necrosis factor receptor (TNFR) superfamily, would be increased and the death inducing signaling complex (DISC) would be detectable. Pregnant rats were injected intracervically with LPS at E15 and immunoblotting, immunohistochemical and immunoprecipitation analyses were performed. The presence of the activated form of the effector caspase (caspase-3) was observed 24 h after LPS administration. Caspase activity assays demonstrated rapid increases in (i) caspases-9 and -10 within 1 h, (ii) caspase-8 at 2 h and (iii) caspase-3 at 4 h. At 24 h after LPS, activated caspase-3(+)/Fas(+) cells were observed within the developing white matter. Lastly, the DISC complex (caspase-8, Fas and Fas-associated death domain (FADD)) was observed within 30 min by immunoprecipitation. Apoptosis in our model occurs via both extrinsic and intrinsic pathways, and activation of Fas may play a role. Understanding the mechanisms of cell death in models of intrauterine inflammation may affect development of future strategies to mitigate these injuries in children.
