ogy & N ol eu ur e ro N p h f y o s l i Matsuo, et al., J Neurol Neurophysiol 2014, 5:3 o a l n o r g u y o J Journal of Neurology & Neurophysiology DOI: 10.4172/2155-9562.1000201 ISSN: 2155-9562 Research Article Open Access Gene Expression Profiling and Bioinformatic Analysis of Rabbit Basilar Artery after Experimental Subarachnoid Hemorrhage Satoshi Matsuo, Yuichiro Kikkawa*, Masaaki Hokama, Ryota Kurogi, Akira Nakamizo, Masahiro Mizoguchi and Tomio Sasaki Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan *Corresponding author: Yuichiro Kikkawa, Department of Neurosurgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, Tel: +81-92-642-5524; Fax: +81-92-642-5526; E-mail: [email protected] Received date: Feb 14, 2014, Accepted date: Mar 20, 2014, Published date: Mar 26, 2014 Copyright: © 2014 Matsuo S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Objective: The molecular mechanisms which contribute to the development of vascular events including cerebral vasospasm after subarachnoid hemorrhage (SAH) in cerebral artery remain to be elucidated. In this study, we investigated the time course of changes in the gene expression of cerebral artery using rabbit SAH model and performed bioinformatic analysis of differentially expressed genes. Methods: Rabbit basilar arteries were harvested on days 3, 5, and 7 after initial hemorrhage. Changes in gene expression of the rabbit basilar artery were investigated by using Agilent rabbit oligo microarrays and analyzed the data by Ingenuity Pathway Analysis (IPA). Results: Among investigated 43,623 genes, 1,121 genes were differentially expressed at least 1 time point. We found that the number, magnitude of fold change, and gene expression pattern were most dynamically changed on day 3, whereas narrowing of the basilar artery became most severe on day 5. In microarray datasets analyzed by IPA revealed that 25 biological functions identified from differentially expressed genes were significantly upregulated. Conclusion: Our findings that were based on gene expression analysis followed by bioinformatic analysis may provide a simple basis to interlink the various presumed pathogenesis of vascular events including cerebral vasospasm after SAH. Keywords: Bioinformatic analysis; Cerebral vasospasm; Gene will produce a broad view of the pathological mechanisms by expression; Ingenuity Pathway Analysis; Microarray; Rabbit model; identifying pathogenic genes and their interaction in various Subarachnoid hemorrhage functional networks. Moreover, analyzing chronological changes in gene expression of cerebral artery may be helpful for elucidating the Introduction mechanism of cerebral vasospasm because the delayed onset is a fundamental feature of the pathophysiology of cerebral vasospasm. Cerebral vasospasm is one of the most important cerebrovascular events following SAH and characterized by delayed and prolonged In this study, we used microarray analysis to investigate contraction of cerebral arteries, which may cause cerebral ischemia chronological changes in gene expression in the basilar artery in the and lead to death or neurological deficits in patients with SAH [1]. rabbit SAH model. Moreover, to elucidate how differentially expressed Therefore, the prevention and treatment of vasospasm have an genes (DEGs) interact during the development of cerebral vasospasm, important role in the management of SAH patients. However, Ingenuity Pathway Analysis (IPA, Ingenuity Systems, and Redwood molecular mechanisms, including alterations in gene expression and City, CA, USA) was performed. functional changes in cerebral arteries after SAH that contribute to the development of cerebral vasospasm, remain to be elucidated. Materials and Methods The gene microarray method allows simultaneous analysis of the expression of thousands of genes. This technology promises to allow Preparation of the rabbit SAH model investigation of the entire genome on a single chip so that researchers This study was conducted in accordance with the guidelines for can obtain a global picture of the interactions among thousands of proper conduct of animal experiments published by the Science genes simultaneously. This technique will also provide insight into the Council of Japan. The study protocol was approved by the Animal underlying mechanisms of many diseases. In some diseases, such as Care and Use Committee, Kyushu University (Permit Number: myocardial infarction and intracranial aneurysm, microarrays have A24-103-0). Adult male Japanese white rabbits (2.5 to 3.0 kg) were been used to elucidate pathological mechanisms by identifying anesthetized with an intramuscular injection of ketamine (40 mg/kg pathogenic genes and their interaction in networks [2-4]. weight) and an intravenous injection of sodium pentobarbital (20 Cerebral vasospasm is thought to be a biologically complex and mg/kg weight). On day 0, 0.5 ml cerebrospinal fluid was aspirated multifactorial pathological condition. Therefore, this new approach percutaneously from the cisterna magna using a 23-gauge-butterfly J Neurol Neurophysiol Volume 5 • Issue 3 • 1000201 ISSN:2155-9562 JNN, an open access journal Citation: Matsuo S, Kikkawa Y, Hokama M, Kurogi R, Nakamizo A, et al. (2014) Gene Expression Profiling and Bioinformatic Analysis of Rabbit Basilar Artery after Experimental Subarachnoid Hemorrhage. J Neurol Neurophysiol 5: 201. doi:10.4172/2155-9562.1000201 Page 2 of 8 needle, and then 2.5 ml of non-heparinized autologous arterial blood quantile algorithm with the ‘preprocessCore’ library package of obtained from the central ear artery was injected over 1 minute. The Bioconductor software [5,6]. To compare the correlation of gene animal was kept in a prone position with the head tilted down at 30° expression among samples at each time point, scatterplots were for 30 minutes. During the animal preparation, blood clot formation created, and the correlation coefficient was calculated using the was not observed in the syringe. On day 2, a second injection of statistical software R version 2.9.0 (http://cran.ism.ac.jp/). Then, we autologous blood was similarly performed. The rabbits which were not identified DEGs in the SAH model using the linear models for subjected to SAH were used as controls (day 0). microarray analysis (limma) package of Bioconductor software [6,7]. Genes in SAH samples (days 3, 5 and 7) with a limma value of P<0.05 Harvest of rabbit basilar arteries and an absolute limma log2 fold-change (|log2 fold change|) higher than 1.0 compared to the control samples (day 0) were defined as On days 3 (n = 3), 5 (n = 3), and 7 (n = 3) after the first hemorrhage, DEGs in this study. Signal intensities of DEGs were log2 transformed, the rabbits were heparinized (400 U/kg weight), euthanized by and hierarchical clustering by both samples and genes was performed intravenous injection of an overdose of sodium pentobarbital (120 and colored by distance from the median value using the Multi mg/kg weight), and exsanguinated from the common carotid artery. Experiment Viewer (MeV) version 4.8.0 (http://www.tm4.org/mev/) Exposing the brain revealed clot formation over the surface of the pons [8,9]. Microarray data are available from the Gene Expression and the basilar artery in the SAH animals (Figure 1A). Immediately Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with the after excising the whole brain en bloc, subarachnoid membrane was accession number GSE44910. In this study, we performed one carefully dissected and the clot was gently removed with micro- microarray analysis per one rabbit because the amount of basilar scissors and micro-forceps not to touch the basilar artery under a artery from each rabbit is too small. binocular microscope (Leica EZ4D, Leica Microsystems, Wetzler, Germany). Distal half of the surface of basilar artery was covered with thick clot in all rabbits with SAH (Figure 1A). Therefore, to estimate vasospasm, the external diameter of the basilar artery was measured at the point of the distal one-third of the rabbit basilar artery. The ventral surface of the whole brain was photographed with a digital camera (CX3, Richo, Tokyo, Japan) and the external diameter of the basilar artery was analyzed withImageJ (National Institute of Health, Bethesda, MD, USA). The entire length of basilar artery was then immediately excised from the brain and dissected free from surrounding tissues with micro-scissors and micro-forceps. Intraluminal blood was gently hand-flushed with normal physiological salt solution (123 mmol/L NaCl, 4.7 mmol/L KCl, 1.25 mmol/L CaCl2 , 1.2 mmol/L MgCl2, 1.2 mmol/L KH2PO4, 15.5 mmol/L NaHCO3, and 11.5 mmol/L D-glucose) using tuberculin syringe, and then the basilar artery was frozen in liquid nitrogen. The frozen tissues were stored at −80°C until use. Basilar arteries harvested from the rabbits which were not subjected to SAH were used as controls (day 0; n = 3). Total RNA isolation from rabbit basilar arteries Total RNA was extracted from the basilar arteries using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocols. The quality of total RNA was evaluated with a spectrophotometer (Nano-Drop2000c; Thermoscientific, Figure 1: Clot formation and time course of narrowing of the rabbit Wilmington, DE, USA) and gel electrophoresis (Experion; Bio-Rad, basilar artery after SAH. (A) Representative photographs of the Hercules, CA, USA). RNA samples with an A260/280 ratio higher than ventral surface of the rabbit brain removed on days 0, 3, 5, and 7 1.8 and with an RNA Quality Index higher than 9.0 was used for the after subarachnoid hemorrhage (SAH). (B) Time course of changes gene expression microarray. Gene expression microarray analysis of in the external diameter of rabbit basilar arteries after SAH.