Establishment and evaluation of inflammation-sensitized hypoxic-ischemic injury in the immature brains

Abstract

Objective \nTo establish a neonatal mouse model mimicking pathological changes of inflammation-sensitized hypoxic-ischemic brain injury in human preterm infants. \n \n \nMethods \nFirst, postnatal day 3 (P3) neonatal mice were randomly assigned into 3 different groups: phosphate buffer (PBS) group (injected intraperitoneally with PBS), lipopolysaccharide (LPS) 0.05 group (injected intraperitoneally with LPS 0.05 mg/kg), and LPS 0.50 mg/kg group (injected intraperitoneally with LPS 0.50 mg/kg). At 24 h after the LPS insult, the weight of mice was measured, and their brains were harvested: 2, 3, 5-triphenyltetrazoulium hydrochloride (TTC) staining was employed for detecting the brain infarct, hematoxylin and eosin (HE) staining for observing neural necrosis, and the terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detecting cell apoptosis. We chose the dosage of LPS with little injury on the neonatal mice for further study. Second, P3 neonatal mice were randomly assigned into 3 different groups: Sham group [without hypox (HI) insult], HI 20 group (exposed to HI for 20 min), and HI 40 group (exposed to HI for 40 min). At 24 h after the HI insult, the weight of mice was measured, and their brains were harvested for TTC staining, HE staining, and TUNEL method. We chose the time of HI with little injury on the neonatal mice for further study. Last, P3 neonatal mice were randomly assigned into 2 different groups: Sham group (PBS injected without HI), and LPS+ HI group (LPS injected 2 hours before HI). We chose the dosage of LPS and the time of HI which had little injury on the neonatal mice for the study. At 24 h after the HI insult, the weight of mice was measured, and their brains were harvested for TTC staining, HE staining, and TUNEL method. \n \n \nResults \nFirst, LPS 0.5 group had lower body weight [(2.63±0.09) g] than both LPS 0.05 group [(2.81±0.14) g] and PBS group [(2.81±0.16) g], so we chose LPS 0.05 mg/kg for further study. Second, HI 40 group had significant brain infarct detected by TTC staining and injured cell detected by HE staining, and TUNEL method showed significant cell apoptosis [(55.67±7.81)%] than in HI 20 group [(2.80±1.74)%], so we chose HI 20 min for further study. Last, LPS+ HI group had lower weight [(2.66±0.09) g] than Sham group [(2.83±0.12) g] (t=2.565, P<0.05), and LPS+ HI group displayed dramatic cell apoptosis [(52.17±9.50)%] than Sham group [(2.15±1.30)%] (t=12.873, P<0.05). \n \n \nConclusion \nThe combination of LPS and HI had significant injury on neonatal mice, which indicates inflammation can sensitize the hypoxic-ischemic injury in the immature brains. \n \n \nKey words: \nPreterm;\xa0Inflammation;\xa0Hypoxia-ischemia;\xa0Brain injury