LPS Sensitizes Hypoxia-ischemia Brain Damage To Newborn Rat

Neonatal hypoxia-ischemia brain damage (HIBD) is one of the most serious diseases, which leads to newborn death and neurological deficit. There is no effective treatment method for HIBD. Recent study showed that perinatal infection plays an important role in neonatal brain damage. To study the effect of infection on the neonatal hypoxia-ischemia brain damage, subclinical dose LPS was used to combine with different time of hypoxia-ischemia (HI). The brain water content, infarct area, TNF- ? and apoptosis inducing factor(AIF) immunohistochemistry(IHC) were used as parameters to evaluate the brain damage and investigate the possible mechanism.Objective:(1) The effect of different LPS dosage: 0.3mg/kg,o.6mg/kg, 0.9mg/kg and control groups, 6 rat pups in each group;(2) The combination effect of LPS and HI to the brain damage in differenttime(10min, 20min, 30min, 40min, 50min): LPS group and control group,30 rat pups in each group. (3) The effect of LPS+HI 20min and HI 55minon the brain infarct area, brain water content, TNF-a and AIFIHC: control group, HI 55min group, LPS+HI 20min group, 34 rat pups in each group.Methods:(D The preparation of the newborn rats HIBD model: At postnatal day (p) 7, pups were anesthetized with inhale ether, the left common carotid artery was ligated and cut. After the surgical procedure, the pups were left to recover for one hour.-The litters were placed in a chamber perfused with humidified air for preheating for 15-30 min and then exposed to a gas mixture (8% oxygen in 92% nitrogen) for 55min. The temperature in the chamber was kept at 37?. After hypoxia, the pups were returned to their dams. Control group only dissected left common carotid artery, no ligature and hypoxia were applied. (2) The preparation of newborn rats LPS+HI model: (D The effect of .different IPS dosage: The pups were injected different dose of LPS (i.p) or saline. The temperature, body weight was measured and serial coronal sections are stained with MAP-2 after 72h. (2) The combination of LPS and HI to brain damage in different time: The pups were ligated with thread and placed in a chamber perfused with 8% oxygen for lOmin, 20min, 30min, 40min, 50min after 4h injection of LPS (0.3mg/kg), the control group only were administered saline. ㏕he preparation of newborn rats LPS+HI model: LPS 0.3mg/kg was administered (i.p) to the 7-day-old rats 4 hours before hypoxia-ischemia, HI group and control group only were administered (i.p)saline. After ligation the left common carotid artery and recovery, the pups were exposed to 8% oxygen gas mixture for 20 min. (3) Measurement of parameters: (D Cerebral infarct size: The rats were decapitated at 3h, 8h, 24h, 72h after hypoxia and the brains were dissected and fixed for 24h with 4% paraformaldehyde in PBS. Serial coronal sections were stained with MAP-2. The MAP-2 negative area was measured with an image-analyzing system. (2) Brain water content: Pups were sacrificed 44h after hypoxia. The olfactorial tubercles, cerebellum and the brainstem were discarded after the brain was dissected out. The hemispheres were separated in the midline and weighted with an accuracy of 10-4g electronic balance and then desiccated at 100? for 5h. Percentage brain water content was determined according to the formula: [(wet weight- dry weight)/wet weight] x 100%. (3) TNF- ? and AIF IHC: Sections were stained with antibodies against TNF-? and AIF, the positive cells were counted in the cortex of MAP-2 negative area with a 400X magnification. Three visual fields were counted and expressed as average number per visual field. (4) Statistical analysis: one way-ANOVA and t test were used and all the data were calculated with SPSS software.Results: (1)LPS+HI model: CD No pups died in LPS o.3mg/kg group and control group. There was no difference between LPS group and control group in temperature and body weight (p>0.05). (2)The mortality increasedwith the prolongation of hypoxia. The brain damage in LPS+HI 20min was much pronounced than t...