The Effect of Early Gestational Intrauterine Infection on the Rat Lung Development and the Possible Mechanisms
|Keywords||intrauterine infection lung development pseudoglandular period cytokines vascular endothelial growth factor surfactant protein|
Objective:The survival of the premature infants and very low birth weight infants is improved as the application of ventilation, surfactant and hormone with the progress of the neonatal intensive care. However, the incidence of premature and the adverse outcome of the premature infant grow up. Intrauterine infection has been proved to play an important role on the lung development. Chorioamnionitis might result in the lung inflammation, induce the expression of the surfactant proteins (SP), improve lung maturation and reduce the incidence of respiratory distress syndrome (RDS). Moveover, the intrauterine infection or inflammation may be a major contributor to the arrested lung development. The "bronchopulmonary dysplasia (BPD)" was defined as oxygen need at 28 days. The pathology of new BPD was marked by impairment in lung alveolarization and aberrant microvasculature, which was associated with inflammation. However, the exact mechanism how intrauterine infection affects the lung growth and development remains unclear. The following factors may contribute to the lung development after intrauterine infection:(1) a large number of proinflammatory cytokines induced by intrauterine infection may induce focal lung inflammation, which may result in lung maturation or injury; (2) intrauterine infection may result in the abnormal vasculature and arrest of alveoli development by downregulating the growth factors involved in the angiogenesis such as vascular endothelial growth factor (VEGF). VEGF is a paracrine mediator, which is abundent in the lung. The biological effects of VEGF are mediated by two receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1 (Flk-1). VEGF could promote the differentiation and proliferation of endothelial cells. VEGF and its receptors are critical to vasculogenesis and alveolarization in the lung development; (3) premature:intrauterine infection is one of the important factors for premature. The premature infants are more likely to suffer with the infection, ventilation and hyperoxia, which will trigger the cascade inflammation reaction, and increase the lung injury.Most researches used the intrauterine infection models by intra-amniotic endotoxin or lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1 injection. Furthermore, in previous experimental studies, intrauterine infection was mainly induced on late lung development stage such as canalicular or saccular stage. Much less is known about the effect of early intrauterine infection at the pseudoglandular stage on the pre and postnatal lung development. This critical stage in lung growth is characterized by fast branching of airway and vascular network and progressive differentiation of epithelial cells. By the end of this period, the formation of conducting airways and terminal bronchioles is complete. Thus, we established a rat model of intrauterine infection during the pseudoglandular period, at embryonic day (E) 15, by inoculating the live bacteria suspension to the uterine cervix.The present study was undertaken to determine whether early intrauterine infection could damage the developing lung. The longitudinal study of the prenatal and postnatal lung morphology alterations, from E17 to postnatal day (P) 14 was performed to investigated effects of maternal inoculation with E.coli on pup’s lung, and we examined alterations in expression of genes implicated in angiogenesis (VEGF, Flt-1 and Flk-1) and surfactant proteins (SP-A and SP-B) in the developing lung. In addition, we also explored the induction of proinflammatory mediators (IL-1βand TNF-α) in pup’s lung after intrauterine E.coli infection.Methods:1. Groups:control group and E.coli-treated group. The pregnant rats at E15 were anesthetized with an intraperitoneal dose of 40 mg/kg body weight of 2% sodium pentobarbital, followed by an endocervical injection of either 0.4 ml of Escherichia coli (E. coli) suspension (n=22) or the same volume of saline (n=22).2. Sample collection:Some of the pregnant rats were caesared at 24 hours, E17, E19 and E21 after endocervical injection, and the fetal lung tissues were collected; others were allowed to deliver and the lung tissues of the neonatal rats at P1, P3, P7 and P14 were collected; the collected lung tissues were fixed in neutral formaldehyde or stored at -80℃freezer.3. Experimental methods:The body weight and lung weight of the pup’s lung were measured during sample collection. The prenatal and postnatal lung morphology were observed by microscopy after hematoxylin-eosin (HE) staining; radical alveolar counts (RAC) was observed, which referred to the number of alveoli transected by a perpendicular line drawn from the center of the respiratory bronchiole to the pleura or the nearest interlobular septum. The expression of IL-1β, TNF-α, VEGF, Flt-1, Flk-1, SP-A and SP-B mRNA in the pup’s lung at E17,19,21 and P1,3,7,14 were quantified by real-time RT-PCR. Western blot or immunohistochemistry analysis were used for evaluation of Flt-1, Flk-1, VEGF and SP-A protein levels in the fetal and neonatal rat lungs.Results1. In the study,44 pregnant rats were inoculated,22 with E.coli (E.coli-treated group) and 22 with saline solution (control group). All the dams had normal food intake and activity, and none of the dams died after inoculation. There were few fetal deaths and pregnant losses after intrauterine infection. Two hundred eight live pups from the control group and two hundred live pups from the E.coli-treated group were eligible for the study. The maternal uterus and placentas of the E.coli-treated group showed signs of diffuse neutrophil infiltrates and there was no evidence of inflammation in the control group rats.2. After treated with E.coli, the body weight was decreased at E17,19,21 and P1, and the lung weight was decreased at E17,19,21, P1 and P3 compared with the control group, while the lung/body weight ratios decreased at E17 and E19 only.3. HE staining revealed that the pup’s lung morphology of the E.coli-treated group was abnormal, with less saccules in the fetal lung and simple alveolarization at the alveolar stage. Especially at P7 and P14, there were fewer alveoli, less small tissue ridges arising from the primary septa in the pups exposure to intrauterine infection. The septa appeared thicker than the controls, in which the septa became thinner during normal alveolar development. The RAC of the E.coli-treated group at P7 and P14 was significantly lower than that of the control group. There was no significant different evidence of alveolar destruction or fibrosis between two groups.4. Following E.coli exposure, the level of IL-1βand TNF-a mRNA in the embryonic lung was similar to the controls. After birth, the level of IL-1βmRNA in the E.coli-treated pup’s lung was increased significantly at P1, P7 and P14, while the level of TNF-a mRNA was upregulated at P1, P3 and P7.5. The levels of SP-A (at E17, E19, E21) and SP-B mRNA (at E19, E21) in the embryonic lung in the E. coli-treated group were greatly lower than that of control group, but there were no differences between the two groups in the postnatal lung, except a decrease of SP-B mRNA in pup’s lung at P7 after E.coli exposure. By immunohistochemistry, we observed an decrease of SP-A at E19 in the embryonic lung in the E.coli-treated group. 6. The fetal lung exposed to E.coli had much lower level of VEGF mRNA at E17 and E19. The Western blot analysis showed that expression of VEGF protein was consistent with the real-time RT-PCR results. Interestingly, we did not observe the significant differences of Flt-1 and Flk-1 between two groups in prenatal lung. The levels of Flt-1 and Flk-1 mRNA were respectively increased at P7, P14 and P1, P7, P14 in E.coli-treated group. By immunohistochemistry, we observed an increase of Flt-1 and Flk-1 at P7 in intrauterine E. coli-treated pup lungs.Conclusion:1. The pathology of the fetal and neonatal lung characterized by fewer alveoli, less septa and thicken septa after maternally endocervical E. coli inoculation, suggest that the early gestational intrauterine E.coli infection might arrest the alveolarization in developing lung, which is similar to the BPD.2. The decrease of SP-A and SP-B expression in the fetal lung indicates that the early gestational intrauterine E.coli infection might fail to accelerate the maturation of fetal lung.3. The increase of TNF-αand IL-1βmRNA levels in the postnatal rat lungs after intrauterine E.coli infection, suggests that the cytokine cascade induced by intrauterine infection may play an important role in the lung injury. The mRNA levels of TNF-a and IL-1βin the fetal rat lungs after intrauterine E.coli infection are similar to the controls, suggesting intrauterine E.coli infection could not induce the increase of proinflammatory cytokines in the fetal lung.4. The disturbed epression of VEGF and its receptors after intrauterine E.coli infection, suggests the mechanism of VEGF signaling may play an important role in the normal lung development.