Tolerance and injury mechanisms in the developing CNS

Abstract

The pathophysiological mechanisms underlying the development of hypoxic-ischemic (HI) brain injury has been the subject of extensive research yet a neuroprotective strategy to limit its progression is lacking.OBJECTIVES: The aim of the present thesis was to obtain a better understanding of the mechanisms involved in neonatal HI, specifically to study I) the involvement of Poly ADP-ribose) polymerase (PARP)-1 in HI, II) the effect of hypoxic PC on brain injury and function after HI in the long-term perspective, III) the effect of hypoxic PC on global gene expression, in particular the involvement of apoptosis related genes, and IV) the vascular response to hypoxic PC.METHODS: The experimental work was based on a perinatal rodent model of unilateral HI. PARP-1 +/+, +/- and -/- mice on a CD-1 background were used in study I, and rats in studies II-IV. Immunohistochemistry and western blotting were used for PAR detection and localization, and colorimetry for measurement of NAD+. For tolerance induction animals were subjected to PC hypoxia prior to HI. Brain injury was assessed using histology and immunohistochemistry. Brain function was evaluated using a forelimb use asymmetry test and the Morris water maze. Global gene expression after PC was studied by using microarray analysis (Affymetrix gene chip rat genome 230 2.0 array). CBF was measured using the iodoantipyrine and laser Doppler techniques. Microvascular density was determined by immunohistochemistry and stereology. RESULTS: I) PARP-1 deficiency conferred moderate protection (p < 0.05) in the total group of animals, but analysis by sex revealed that males, but not females, were protected (p < 0.05). PARP-1 was activated 1 24 h after HI in neonatal +/+ mice and rats of both sexes, whereas the decrease of NAD+ during the early post-HI phase was only seen in males. II) Neuropathological evaluations at eight weeks post-HI demonstrated substantial protection in PC animals as compared to sham. Furthermore, the performance in the functional tests 6-8 weeks after HI was greatly improved in PC rats; III) Hypoxic PC induced >1000 genes which could be involved in development of tolerance and 77 of these transcripts were related to apoptosis. IV) The decrease in cortical CBF during HI was attenuated after hypoxic PC. Modulation of the CBF was not dependent on production of NO. Numerous vascular related genes were induced after PC, primarily involved in vasoregulation and angiogenesis. PC induced remodeling of the microvascular structure resulting in a significant increase in the vascular density 24h after PC. CONCLUSIONS: I) These results, indicate that HI activates PARP-1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury. II) Induction of hypoxic PC prior to HI provides long-lasting neurological as well as functional protection in the immature rat. III) PC influences the global gene expression including multiple genes related to the apoptotic cascade. IV) PC affects vascular related genes and increases the microvascular density which partly could explain the attenuated decrease of CBF during subsequent HI and the reduction of brain injury.