The Associated Effect Of Intrauterine Infection And Interuterine Asphyxia To Premature Rat's Brain Damage

Large numbers of researches have proved that both intrauterine infection and intrauterine asphyxia can induce premature brain damage and they are the main reasons of it. But intrauterine infection and intrauterine asphyxia coexist in many clinical cases, while researches about whether can they aggravate the degree of premature brain damage by coexistence are not found both at home and at board. So, we will research the associated effect of them to premature brain damage by upbuild the animal model, and farther investigate the pathogenesis through measure the expression of glial fibrillary acidic protein(GFAP) tumor necrosis factor- α (TNF-α) tissue-type plasminogen Activator tPA and neuron apoptosis at different time points by immunohistochemistry, in order to offer theoretic advice to prevention and cure to premature brain damage.Objiects:Pregnant rats on gestation day 18 were randomly divided into two teams. The first team was separated into four groups: 1. NS+sham operation; 2. LPS (O55: B5, Sigma, 1mg) ; 3. intrauterine asphyxia; 4. LPS + intrauterine asphyxia. Every group was 6 pregnant rats, the fetal rats' brains were taken out by laparotomy after 72h of different disposal. The second team was separated into there groups: 1.NS+shamoperation; 2. NS+ intrauterine asphyxia; 3. LPS + intrauterine asphyxia. Every group was 6 pregnant rats, they were all executed 4h 12h 24h 48h 72h after operation respectively, brains of the fatal rats were taken out by laparotomy.Methods:l.Pregnancy rat: Adult male and female SD rats were bred in the same cage in proportion of 4:1, vaginal secretion were smeared every morning at 8 from the second day. The first day of pregnancy was made sure by found vaginal embolus or spermatozoa all the visual field under optical microscope.2.Premature animal model of intrauterine infection and intrauterine asphyxia: Pregnant rats on gestation day 18 underwent sterile surgery four hours after intra-abdominal inject LPS 0.5mg/kg, they were under aether anaesthesia , fixed resupine upon the operating table, and midline abdominal incision were performed. The uterus was pulled out and placed upon wet pledget carefully, then the uterus arterial and ramus vascular of placenta were separated and ligated for 15min. After that, wet and warm pledget were covered upon the uterus to maintain the temperature and humidity. When intrauterine asphyxia was finished, the uterus was lay back into the celiac, and the wound was sutured and bound up.While the uterus arterial and ramus vascular of NS+ sham operation pregnant rats were only sepatated but not ligated four hours after intra-abdominal inject NS lml, which of NS+ asphyxia pregnant rats were sepatated and ligated 15mins four hours after intra-abdominal inject NS lml, the methods was same to above. 3.The methods to induce premature rat brain damage by intrauterine infection associate with intrauterine asphyxia is make sure to be intra-abdominal inject LPS 0.5mg/kg + intrauterine asphyxia 15min though advance experiments. 4. Specimen: The pups were taken out by laparotomy and executed at different time point after operation, After anaesthesia by aether, they were perfused intracardially with NS and 10% buffered formaldehyde till the bodies became paleness and stiff. Brains were taken out and fixed up in 10% buffered formaldehyde at least 72h, then embedded by paraffin after dehydration and vitrification. Consecutive coronalsections of hippocampus at 5 n m of thickness were prepared.5.Histological evaluation: The sections were given HE coloration andimmunohistochemistry of TUNEL> GE/U\ TNF-a ? tPA respectively.Results:1. HE: Compared with group LPS and NS + sham operation, the level of brain cell edema and the disorganization of group LPS + asphyxia are more serious, and the number of apoptosis cell are more large. Besides, group LPS + asphyxia have haemorrhage of dorsal corner of ventricle > enlargment of ventricle and cyst of dorsal corner of ventricle, while group asphyxia don't have these phenomena, and group LPS only have 1 case of haemorrhage of dorsal corner of ventricle.2. TUNEL and GFAP : They have the same results: The number of positive cell is group LPS + asphyxia> group LPS^ group asphyxia> group NS + sham operation. There is significant difference among group LPS + asphyxia and group LPS^ group asphyxia ^ group NS + sham operation (p<0.05). There is also significant difference between group LPS or group asphyxia and NS + sham operation (p<0.05). But there is no significant difference between group LPS and group asphyxia (p>0.05) .3. TNF- a : There is significent difference of the number of positive cell among group NS + sham operation > group asphyxia and group LPS + asphyxia 4h after operation (p <0.001) . The number of positive cell goes the peak 12h after operation and begin to reduce after that, but there is still significent difference among the three groups till 24h after operation (p<0.001) . There is no significent difference between group asphyxia and NS + sham operation (p>0.05) but between LPS + asphyxia and NS + sham operation (p<0.001) 48h after operation. There is no difference among the three groups 72h after operation (p>0.05) .4. tPA: There is no significent difference of the number of positive cell among group NS + sham operation > group asphyxia and group LPS + asphyxia 4h after operation (p >0.05) . 12h after operation there is significent difference among the three groups (p <0.001) , and this keep on to 24h (p<0.05) after operation when the number goes to peak. Then the number begin to reduce, but there is still significent difference amongthe three groups till 48h after operation (p<0.05) . There is no significent difference between group asphyxia and group NS + sham operation (p>0.05) but between LPS + asphyxia and NS + sham operation (p<0.001) 72h after operation.Conclusion:1. Both intrauterine infection and intrauterine asphyxia can induce premature rat brain damage.2. The association of intrauterine infection and intrauterine asphyxia can aggravate the degree of premature brain damage.3. Apoptosis> TNF- a and tPA take part in the pathogenesis of hypoxic-ischamic brain damage.