Project description:Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, and results in brain tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, and mechanisms therein. However, before proceeding to the neuroprotection aspect of our research, we initiated a study with a primary aim to investigate the molecular responses at the level of gene expression in ischemic brain tissue. To do so, we used permanent middle cerebral artery occlusion (PMCAO) model mice in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, saline injected mice brain in sham (control, n=3) and PMCAO (treatment, n=3) mice were dissected into left (contralateral) and right hemispheres (ipsilateral) at two time points, 6 and 24 h after injection. The ipsilateral hemisphere shows ischemia, within 24 h, and is marked by cell death as visualized by TTC staining. Tissues were ground into fine powder with liquid nitrogen and total RNA was extracted, followed by quality check using gel electrophoresis and cDNA synthesis in conjunction with RT-PCR using certain marker genes. The obtained good quality total RNA from ipsilateral hemisphere was used for DNA microarray analysis on a mouse (Mus musculus) whole genome 4x44K DNA chip by using the dye-swap approach. Results revealed a large number of changed gene expressions at both 6 (1237 up- and 620 down-regulated) and 24 h (2759 up- and 2102 down-regulated). 792 and 167 genes were found to be commonly up- and down-regulated 6 and 24 h post-ischemia, respectively. Functional categorization using the gene ontology (GO, MGD/AMIGO) of these gene expressions revealed major categories of cellular processes, biological regulation, regulation of biological processes, metabolic processes, and response to stimulus. In addition, RT-PCR using specific primers of randomly selected genes was used to validate the changed gene expressions. This study provides the first inventory of ischemia-related transcriptome in mouse brain. For appropriate control to the PMCAO model, mice were anesthetized with 4% isoflurane (induction) and 2% isoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An incision was then made in the cervical skin followed by opening of salivary gland, and visualization of the right common carotid artery. The external carotid artery was exposed through a midline cervical incision, and subsequently, the wound was closed by sutures. For the PMCAO model, we used the intraluminal filament technique, where a 7-0 monofilament nylon suture with its tip slightly rounded by heating was inserted into the common carotid artery followed by placement into the middle cerebral artery. In both the control and PMCAO model, saline (0.9% NaCl) was injected into the intracerebroventricle, and the animals were returned to their cages. A total of eight groups were prepared with three mice each in the control (4 groups) and PMCAO model (4 groups). After 6 and 24 h post-injection of saline, mice were removed from the cages and decapitated using scissors, and the whole brains were carefully dissected on ice. The left (contralateral) and right (ipsilateral) hemispheres were separated and placed in 2 mL Eppendorf tubes followed by quickly immersing the tubes in liquid nitrogen (Lq. N2), and then stored in -80ÂºC prior to further analysis. A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis using the contralateral hemispheres. The effects of ischemia were checked over the SHAM control mice at 6 and 24 h post-saline injection.

Project description:The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. - This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding sham control that used 0.9% saline injection. Ischemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. - Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expresison using DNA microarray chip (4x44K, Agilent) and one-/two-dimensional gel electrophoresis (1-/2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. - Our results revealed numerous changes in the transcriptome of ischemic (ipsilateral) hemisphere treated with PACAP38 over the saline-injected SHAM control hemisphere. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic region that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the activated neuronal defense mechanisms, and interestingly, PACAP strongly suppresed this induction. - This study provides the first inventory of PACAP influenced gene and protein targets in ischemic hemisphere, and provides new targets for thereaupetic interventions. The PMCAO model mice were generated as described previously. Briefly, the mice were anesthetized with 4% sevoflurane (induction) and 2% sevoflurane (maintenance) in a 30% O2 and 70% N2O gas mixture via a face mask. An incision was then made in the cervical skin followed by opening of the salivary gland, and the right common carotid artery was visualized. A midline cervical incision was made to expose the external carotid artery. The intraluminal filament technique was used, to generate the PMCAO model. - The PACAP38 (1 M-BM-5l containing 1 pmol) or 1 M-BM-5l of saline (0.9% NaCl) was injected intracerebroventrically, immediately after PMCAO. PACAP38 (Peptide Institute Inc., Osaka, Japan; supplier temperature was -20M-BM-:C and was stored at -80M-BM-:C) was dissolved in, and diluted M-CM-^W10 times with 0.9% NaCl just before use. - In the sham control animals, following exposure of the external carotid artery, the wound was sutured. The PACAP38 or saline was injected intracerebroventrically in the same concentrations as above. After injections, the animals were returned to their cages. - A total of eight groups were prepared: four groups of three, seven, four and five mice in the PMCAO plus PACAP38 and PMCAO plus saline cohorts at 6 and 24 h after operation, respectively, and five mice each in the control (sham) plus PACAP and saline groups at 6 and 24 h after operation, respectively. - We used three mice each in PMCAO groups that exhibited neurological grades G1 and G2 and three mice each at random in sham groups for the subsequent downstream analysis. Six or 24 hours post-injection of PACAP38 or saline, the mice were removed from their cages, decapitated, and their brains carefully removed on ice. The left (contralateral) and right (ipsilateral; ischemic) hemispheres were dissected and placed in 2 mL Eppendorf tubes, which were then quickly immersed in liquid nitrogen before being stored in -80M-BM-:C prior to further analysis. - For analysis of gene components, the tissues were ground to a very fine powder with liquid nitrogen, used for total RNA extraction, quantity and quality check, followed by cDNA synthesis, and RT-PCR. A mouse 4 x 44K whole genome oligo DNA microarray chip (G4122F, Agilent Technologies, Palo Alto, CA, USA) was used for global gene expression analysis using the ipsilateral (ischemic) hemisphere. Briefly, total RNA (900 ng; 300 ng each replicate pooled together) was labeled with either Cy3 or Cy5 dye using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Fluorescently labeled targets of control (sham) as well as treated (PMCAO) samples with PACAP38 or without PACAP38 (saline) were hybridized to the same microarray slide with 60-mer probes. - Here, we compared the PMCAO plus PACAP38 injected mice to PMCAO plus saline, i.e. the ipsilateral brain region of the PMCAO mice was compared with the same right hemisphere of the control mice. Similarly, Sham control plus PACAP38 injected mice to Sham control plus saline were analyzed to identify only the PACAP38 influenced genes. A flip labeling (dye-swap or reverse labeling with Cy3 and Cy5 dyes) procedure was followed to nullify the dye bias associated with unequal incorporation of the two Cy dyes into cDNA.

Project description:Brain ischemia, also termed cerebral ischemia, is a condition in which there is insufficient blood flow to the brain to meet metabolic demand, and results in brain tissue death (cerebral infarction) due to poor oxygen supply (cerebral hypoxia). Our group is interested in the protective effects of neuropeptides for alleviating brain ischemia, and mechanisms therein. However, before proceeding to the neuroprotection aspect of our research, we initiated a study with a primary aim to investigate the molecular responses at the level of gene expression in ischemic brain tissue. To do so, we used permanent middle cerebral artery occlusion (PMCAO) model mice in combination with high-throughput DNA microarray analysis on an Agilent microarray platform. Briefly, saline injected mice brain in sham (control, n=3) and PMCAO (treatment, n=3) mice were dissected into left (contralateral) and right hemispheres (ipsilateral) at two time points, 6 and 24 h after injection. The ipsilateral hemisphere shows ischemia, within 24 h, and is marked by cell death as visualized by TTC staining. Tissues were ground into fine powder with liquid nitrogen and total RNA was extracted, followed by quality check using gel electrophoresis and cDNA synthesis in conjunction with RT-PCR using certain marker genes. The obtained good quality total RNA from ipsilateral hemisphere was used for DNA microarray analysis on a mouse (Mus musculus) whole genome 4x44K DNA chip by using the dye-swap approach. Results revealed a large number of changed gene expressions at both 6 (1237 up- and 620 down-regulated) and 24 h (2759 up- and 2102 down-regulated). 792 and 167 genes were found to be commonly up- and down-regulated 6 and 24 h post-ischemia, respectively. Functional categorization using the gene ontology (GO, MGD/AMIGO) of these gene expressions revealed major categories of cellular processes, biological regulation, regulation of biological processes, metabolic processes, and response to stimulus. In addition, RT-PCR using specific primers of randomly selected genes was used to validate the changed gene expressions. This study provides the first inventory of ischemia-related transcriptome in mouse brain.

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MRNA profiles after experimental ischemia

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