ATTIVAZIONE PRECOCE DEL PROCESSO INFIAMMATORIO IN UN MODELLO SPERIMENTALE DI EMORRAGIA SUBARACNOIDEA

Early activation of inflammation in an experimental model of subarachnoid hemorrhage Introduction. There is increasing evidence that inflammation plays a critical role in the pathogenesis of vasospasm and delayed ischemic deficits after Subarachnoid Hemorrhage (SAH). Objective. Aim of this study is to evaluate the early activation of inflammatory response at the level of basilar artery in a model of single hemorrhage in rats. Material and Methods. A single hemorrhage model was used. CD-IGS rats (250-350 gr) were anesthetized using an i.p. administration of ketamine (100mg/Kg) and midazolam (1 mg/Kg) and subcutaneous fentanyl (0.0075 mg) to control pain. In SAH group, subarachnoid hemorrhage was induced by injection of autologous arterial blood into the cisterna magna of the animal throughout a suboccipital puncture (200 µl in 3 min). Animals were therefore maintained with the head down for 15 minutes and were leaved to breath autonomously and to recover. In Sham group, rats were submitted to the same procedures, puncturing the cisterna magna, without injecting arterial blood in it. In basal group, rats were not submitted to any surgical procedure. In short term experiments, animals were sacrificed 4 hours after the puncture. Basilar artery was then removed from the rat brain and freezed. A pool of 3 arteries were necessary in order to obtain adequate amount of RNA to perform gene expression analysis. 4 pool of arteries for each group was submitted to a real-time PCR analysis to evaluate expression of the following genes: TNF-α, IL-6, CXCL-1, iNOS, ET-1, ICAM-1. 3 pools of arteries for each group were submitted to TaqMan Low Density Array. TaqMan Low Density Array is a consumable consisting of 384 wells connected by a series of microfluidic channels, designed to be used on the Applied Biosystems 7900HT Fast Real-Time PCR System platform. Microfluidic technology allows multiple targets to be analyzed across many samples simultaneously. These arrays are used to analyze specific biological pathway activation and the expression of genes of interest. In this work, 90 genes of interest related to the inflammatory response, oxidative stress, repair mechanisms and apoptosis, and 7 genes of reference were analyzed. In long term experiments, rats were divided in 2 groups: SAH group and basal group. In SAH group, animals were sacrificed 5 days after procedure or at the occurrence of any clinical disturbances. At sacrifice animals were perfused with isotonic phosphate-buffered saline (PBS) and fixed with paraformaldehyde. Brain was therefore removed and freezed in a solution of OCT, isopentane and liquid nitrogen. Section of 20 m were performed in the brain stem of the rats, in order to complete a morphometric analysis (diameter, area, thickness) of the basilar artery at three levels. Results. Preliminary analysis of genes expression with RT-PCR showed the early up-regulation of all genes in the SAH group: TNF-α (SAH 3.2, SHAM 2.2, CONTROL 1.1), IL-6 (SAH 343, SHAM 2, CONTROL 1); CXCL-1 (SAH 187, SHAM 42, CONTROL 1), iNOS (SAH 39, SHAM 2, CONTROL 0), ET-1 (SAH 2.6, SHAM 1.2, CONTROL 1.1), ICAM-1 (SAH 5.1, SHAM 1.8, CONTROL 1.1). Subsequent analysis with Taqman Low Density array allowed to confirm the clear differentiation of the SAH group with respect to the basal and SHAM groups (p=0.0370). With the assistance of the software EASE, the analysis was deepened in order to evaluate the hypothetic modulation of molecular processes in SAH group. This demonstrated an up-regulation of genes involved in different aspects of immune response, response to stress or external stimulus, and tissue repair, while genes coding for structural protein of intercellular junction or localized at the level of cell membrane are down-regulated. Morphometric analysis in long-term experiments showed the reduction of basilar artery diameter and area in the SAH group (55 ± 15 microm vs 91 ± 20 microm and 1381 ± 150 microm2 vs 3898± 214 microm2). Conclusions These results show an early expression of genes involved in inflammatory process on the wall of the intracranial vessels exposed to subarachnoid blood injection in a model of single hemorrhage in rats. This seems to confirm that inflammation plays a crucial role in the cascade of events after SAH, leading to vasospasm and delayed ischemic deficits, and suggests that new treatments capable to modulate this response could eventually be beneficial.