Inflammatory response in the immature brainafter hypoxia-ischemia. Application of microarray and transgenic technology

Abstract: Hypoxic-ischemic (HI) brain injury remains a common problem encountered during theneonatal period, and it is a major cause of perinatal mortality and long term neurologicalimpairments. After HI, an inflammatory reaction is elicited in the brain, which is believedto contribute to the secondary progression of brain injury.The aim of this thesis was to investigate the inflammatory response and its possiblepathophysiological role after HI in the immature brain.Using a model of cerebral HI in 7-day-old rats, the expression of caspase-1 and IL-18 wasinvestigated by RT-PCR, Western blot and immunohistochemistry and the expression ofIL-1beta by ELISA and immunohistochemistry at various time points after HI. Theinvolvement of different members of the IL-1 family in the development of HI injury wasinvestigated in 9-day-old mice by using different transgenic mice and comparing theirsusceptibility to HI injury with wild-type mice. Global changes in gene expression atvarious time points after HI was investigated with Affymetrix Gene Chips (MG-U74Av2).After HI, caspase-1 and IL-18 mRNA and protein were increased in a similar pattern,starting at 1 day of reperfusion and reaching maximal levels 14 days after the insult. IL-1betawas expressed maximally at 8h after HI, when a 7-fold increase was detected comparedwith contralateral hemispheres. IL-18 deficient mice showed reduced injury after HIcompared with wild-type mice in both gray (-21% overall injury) and white matter(remaining amount MBP and NF was 92% and 78% higher, respectively, in IL-18-deficient compared with control mice). Mice deficient for IL-1beta or the combination ofIL-1alfabeta showed no reduced injury after HI compared with wild-type. When studyingchanges in global gene expression, a total of 491 genes were differentially expressed 2h to72h after HI, using the Significance analysis of microarray (SAM) statistical program withthe following criteria (FDR <10%, fold change 1.5). More than 90% of the differentiallyexpressed genes were previously unreported after HI in the immature brain. Real-timePCR confirmed the expression of 11 upregulated genes. Of the differentially expressedgenes, 29% belonged to the group of immune-inflammatory related genes. Our dataindicate that microglia/macrophages, T- and B- cells, NK-cells, mast cells, dendritic cells,and polymorphonuclear leukocytes may participate in the response to HI. In addition,novel cytokines/chemokines, complement-related, IFN-regulated, components of theTIR/NF-kB pathway and a number of immuno-modulatory genes were induced.In conclusion, the results from this thesis indicate that IL-18 is involved in HI injury inboth gray and white matter, while IL-1 does not seem to be a contributor. In addition,this study has provided novel information about changes in inflammatory gene expressionafter HI, and several inflammatory mediators which may be of pathophysiologicalsignificance after neonatal HI have been identified.