gender: male age: 15-20 weeks weight: 3 to 4 kg tissue: Total heart (atria and ventricles) treatment: Cardioplegia
Treatment protocol
In situ blood perfused cardiopulmonary bypass (CPB): Rabbits were sedated with acepromazine (0.75 mg/kg IM (intramuscular)) and a 21-gage butterfly catheter was placed in the ear and looped and secured in place with tape and vet wrap. Ketamine (35 mg/kg) and xylazine (2.5 mg/kg) IV and were administered via the catheter which was also used intra-operatively to administer heparin (3 mg/kg IV), lidocaine (1% solution, 5 ml) and Lactated Ringer’s solution (10 mL/kg/hr). The surgical site was shaved and prepped with Betadine solution and 70% isopropyl alcohol, each applied in triplicate and patted dry with sterile gauze pads and the entire animal was draped with sterile towels (except for the surgical sites). The larynx was anesthetized with a 1% lidocaine solution and the rabbit was intubated with an uncuffed endotracheal tube (pediatric size 2-0 or 3-0 ID ) and the rabbit was placed on mechanical ventilation (oxygen 40%; tidal volume = 18 ml/kg; ventilation rate 16-18 breaths/min). Proper endotracheal tube placement was verified by auscultation and observation of condensation of the end of the tube. General anesthesia was induced with 3.0% isoflurane and maintained at 1.5% for the duration of the surgical procedure. The animal was secured on the operating room table using soft restraints. Cardiopulmonary Bypass: A medial sternotomy was performed and the heart was exposed and the pericardial sac opened. The cardiopulmonary bypass circuit was achieved using a Prolene (6-0) cardiovascular suture to form a single purse-string layer in the ascending aorta. An incision was made within the purse-string to allow insertion of an aortic cannula (3.3 mm). The cannula was secured with a purse-string tourniquet. A 14 Fr venous cannula was inserted in the right atrium via the auricular appendage. The cardiopulmonary circuit consisted of a roller pump (American Optical Corp., Southbridge, MA) and a neonate membrane oxygenator (Sorin Group USA, St. Louis, MO). The circuit was primed with whole blood obtained from a donor rabbit. During cardiopulmonary bypass when oxygenation was no longer occurring via ventilator, propofol (0.5 – 0.7 mg/kg/min IV) was continuously infused via the marginal ear vein. Blood Donor Rabbits Donor rabbits were sedated and anesthetized as described above. A medial sternotomy was performed and the heart was exposed, a large bore needle was inserted into the heart and blood was withdrawn into a 60 ml syringe. The heart was then removed and the animal died by exsanguination. The blood was collected in blood bags containing citric acid (Baxter, Deerfield, IL) and used immediately to prime the cardiopulmonary bypass circuit . The blood volume in the priming solution was calculated for the final hematocrit of the priming volume to be 18-20%. Mannitol (15%) and sodium bicarbonate (20 mEq/1) were added for pH adjustment. Blood gases and hematocrit were monitored every 10-15 minutes using a Corning 238 pH/blood gas analyzer and a Corning 270 CO-oximeter (Chiron Diagnostics, Emeryville, CA). Treatment Groups: Three treatment groups were investigated. Control treatment animals were sedated, anesthetized and received a sternotomy; the aorta, right atrium\auricular appendage and the apex were sham-manipulated in the same manner as for global ischemia and cardioplegia hearts. Control hearts received no ischemia or cardioplegia. Control hearts were removed from the animal following 180 minutes (30 minutes equilibrium, 30 minutes sham ischemia and 120 minutes sham reperfusion). Global ischemia hearts received 30 minutes of global ischemia. Global ischemia was achieved by crossclamping of the aorta. Cardioplegia hearts received cold blood cardioplegia (Deaconess Surgical Associates (DSA) solution (K+, 60 mM; MgSO4 , 8 mM; dextrose, 2.5 mM; THAM (tris(hydroxymethyl) aminomethane), 10 mM in normal (0.9%) saline and 50 uM diazoxide) mixed (1:4; v:v) with cold blood). Diazoxide (DZX) was dissolved in dimethyl sulfoxide (DMSO, Fisher Scientific Co., Fair Lawn, NJ) before being added to DSA cardioplegia. The final concentration of DMSO was less than 0.1%. DMSO was added to control and global ischemia hearts at the same concentration. Cardioplegia was administered antegradely through the aortic root (20 ml/kg) to induce cardiac arrest. Following 30 minutes of cardioplegia the crossclamp was released, the cardiopulmonary bypass was ceased and the hearts were reperfused for 120 minutes. Langendorff Perfusion: To ensure that the heart tissue did not contain blood, a possible source of contamination in subsequent molecular isolation and analysis, following 120 minutes of reperfusion the heart was excised from the animal and placed in an ice-cold bath of 4oC Krebs-Ringer solution containing; NaCl 100 mM, KCl 4.7 mM, KH2PO4 1.1 mM, MgSO4 1.2 mM, NaHCO3 25 mM, CaCl2 1.7 mM, glucose 11.5 mM, pyruvic acid 4.9 mM, and fumaric acid 5.4 mM and equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37oC), where spontaneous beating ceased within a few seconds. The aorta was cannulated with a polyethylene cannula, and the heart was subjected to Langendorff retrograde perfusion for 10 minutes through the aortic root with Krebs-Ringer solution to remove all blood. Langendorff perfusion was performed at a constant pressure of 75 cm H2O in a water-jacketed chamber with myocardial temperature maintained at 37oC. The blood free heart was removed from perfusion and the atria and large vessels removed. The heart was then quick frozen in liquid nitrogen and then stored in liquid nitrogen prior to the isolation of myocardial RNA and proteins. RNA and Protein Isolation: Total tissue RNA was isolated and quality and purity was assessed by spectrophotometric analysis and agarose gel electrophoresis. Total tissue ventricular myocardial protein was isolated and quality and purity was assessed by poly-acrylamide gel electrophoresis.
Extracted molecule
total RNA
Extraction protocol
Total heart (atria and ventricles) RNA extracted using Trizol following manufacturer's instructions
Label
Cy3
Label protocol
Total RNA was isolated separately from Control, Global ischemia and Cardioplegia treated New Zealnad White Rabbit heart tissue using Trizol reagent (Gibco BRL- Life Technologies), according to the manufacturer’s protocol. Integrity of the RNA was determined by 1% agarose gel containing 3% formaldehyde, 0.02 M MOPS pH 7.4 (3-[N-morpholino] propanesulfonic acid) and 0.001 M disodium EDTA (ethylenediamine tetraacetic acid).RNA (35 ug) from control and experimental groups was used separately for first strand cDNA synthesis using an oligo-dT primer (0.5ug /uL). Following incubation at 65 oC for 5 min. the reaction was put on ice and the following added: 4uL first strand buffer; 2uL 0.1M DTT; 2uL 10X low dTTP–dNTP (5 mM dATP, dCTP, dGTP, and 2 mM dTTP); 2uL CyanineX-dUTP (1 mM ; Cyanine3; Cy3 for control; green and Cyanine5; Cy5 for experimental; Amersham, Pharmacia Biotech); 1uL RNasin and 1uL of Superscript II (GIBCO) and incubated at 42 oC for 30 min. then and another 1uL Superscript II added and the reaction allowed to continue a further 40 min. at 42 oC. The reaction was stopped with the addition of 2.5 uL 500 mM EDTA and heating to 65 oC for 1 min. The RNA template was hydrolyzed with the addition of 5 uL 1M NaOH and incubation at 65 oC for 10 min. and then neutralized by adding 12.5 uL 1M Tris pH 7.5 immediately to neutralize the pH. The labeled cDNA probes were purified by gel exclusion chromatography (ProbeQuant G-50, Amersham) and the volume reduced to 5-10 uL using a Speedvac.
Channel 2
Source name
New Zealand White rabbits (male, 15-20 weeks; 3 to 4 kg)
gender: male age: 15-20 weeks weight: 3 to 4 kg tissue: Total heart (atria and ventricles) treatment: Global Ischemia
Treatment protocol
In situ blood perfused cardiopulmonary bypass (CPB): Rabbits were sedated with acepromazine (0.75 mg/kg IM (intramuscular)) and a 21-gage butterfly catheter was placed in the ear and looped and secured in place with tape and vet wrap. Ketamine (35 mg/kg) and xylazine (2.5 mg/kg) IV and were administered via the catheter which was also used intra-operatively to administer heparin (3 mg/kg IV), lidocaine (1% solution, 5 ml) and Lactated Ringer’s solution (10 mL/kg/hr). The surgical site was shaved and prepped with Betadine solution and 70% isopropyl alcohol, each applied in triplicate and patted dry with sterile gauze pads and the entire animal was draped with sterile towels (except for the surgical sites). The larynx was anesthetized with a 1% lidocaine solution and the rabbit was intubated with an uncuffed endotracheal tube (pediatric size 2-0 or 3-0 ID ) and the rabbit was placed on mechanical ventilation (oxygen 40%; tidal volume = 18 ml/kg; ventilation rate 16-18 breaths/min). Proper endotracheal tube placement was verified by auscultation and observation of condensation of the end of the tube. General anesthesia was induced with 3.0% isoflurane and maintained at 1.5% for the duration of the surgical procedure. The animal was secured on the operating room table using soft restraints. Cardiopulmonary Bypass: A medial sternotomy was performed and the heart was exposed and the pericardial sac opened. The cardiopulmonary bypass circuit was achieved using a Prolene (6-0) cardiovascular suture to form a single purse-string layer in the ascending aorta. An incision was made within the purse-string to allow insertion of an aortic cannula (3.3 mm). The cannula was secured with a purse-string tourniquet. A 14 Fr venous cannula was inserted in the right atrium via the auricular appendage. The cardiopulmonary circuit consisted of a roller pump (American Optical Corp., Southbridge, MA) and a neonate membrane oxygenator (Sorin Group USA, St. Louis, MO). The circuit was primed with whole blood obtained from a donor rabbit. During cardiopulmonary bypass when oxygenation was no longer occurring via ventilator, propofol (0.5 – 0.7 mg/kg/min IV) was continuously infused via the marginal ear vein. Blood Donor Rabbits Donor rabbits were sedated and anesthetized as described above. A medial sternotomy was performed and the heart was exposed, a large bore needle was inserted into the heart and blood was withdrawn into a 60 ml syringe. The heart was then removed and the animal died by exsanguination. The blood was collected in blood bags containing citric acid (Baxter, Deerfield, IL) and used immediately to prime the cardiopulmonary bypass circuit . The blood volume in the priming solution was calculated for the final hematocrit of the priming volume to be 18-20%. Mannitol (15%) and sodium bicarbonate (20 mEq/1) were added for pH adjustment. Blood gases and hematocrit were monitored every 10-15 minutes using a Corning 238 pH/blood gas analyzer and a Corning 270 CO-oximeter (Chiron Diagnostics, Emeryville, CA). Treatment Groups: Three treatment groups were investigated. Control treatment animals were sedated, anesthetized and received a sternotomy; the aorta, right atrium\auricular appendage and the apex were sham-manipulated in the same manner as for global ischemia and cardioplegia hearts. Control hearts received no ischemia or cardioplegia. Control hearts were removed from the animal following 180 minutes (30 minutes equilibrium, 30 minutes sham ischemia and 120 minutes sham reperfusion). Global ischemia hearts received 30 minutes of global ischemia. Global ischemia was achieved by crossclamping of the aorta. Cardioplegia hearts received cold blood cardioplegia (Deaconess Surgical Associates (DSA) solution (K+, 60 mM; MgSO4 , 8 mM; dextrose, 2.5 mM; THAM (tris(hydroxymethyl) aminomethane), 10 mM in normal (0.9%) saline and 50 uM diazoxide) mixed (1:4; v:v) with cold blood). Diazoxide (DZX) was dissolved in dimethyl sulfoxide (DMSO, Fisher Scientific Co., Fair Lawn, NJ) before being added to DSA cardioplegia. The final concentration of DMSO was less than 0.1%. DMSO was added to control and global ischemia hearts at the same concentration. Cardioplegia was administered antegradely through the aortic root (20 ml/kg) to induce cardiac arrest. Following 30 minutes of cardioplegia the crossclamp was released, the cardiopulmonary bypass was ceased and the hearts were reperfused for 120 minutes. Langendorff Perfusion: To ensure that the heart tissue did not contain blood, a possible source of contamination in subsequent molecular isolation and analysis, following 120 minutes of reperfusion the heart was excised from the animal and placed in an ice-cold bath of 4oC Krebs-Ringer solution containing; NaCl 100 mM, KCl 4.7 mM, KH2PO4 1.1 mM, MgSO4 1.2 mM, NaHCO3 25 mM, CaCl2 1.7 mM, glucose 11.5 mM, pyruvic acid 4.9 mM, and fumaric acid 5.4 mM and equilibrated with 95% O2 and 5% CO2 (pH 7.4 at 37oC), where spontaneous beating ceased within a few seconds. The aorta was cannulated with a polyethylene cannula, and the heart was subjected to Langendorff retrograde perfusion for 10 minutes through the aortic root with Krebs-Ringer solution to remove all blood. Langendorff perfusion was performed at a constant pressure of 75 cm H2O in a water-jacketed chamber with myocardial temperature maintained at 37oC. The blood free heart was removed from perfusion and the atria and large vessels removed. The heart was then quick frozen in liquid nitrogen and then stored in liquid nitrogen prior to the isolation of myocardial RNA and proteins. RNA and Protein Isolation: Total tissue RNA was isolated and quality and purity was assessed by spectrophotometric analysis and agarose gel electrophoresis. Total tissue ventricular myocardial protein was isolated and quality and purity was assessed by poly-acrylamide gel electrophoresis.
Extracted molecule
total RNA
Extraction protocol
Total heart (atria and ventricles) RNA extracted using Trizol following manufacturer's instructions
Label
Cy5
Label protocol
Total RNA was isolated separately from Control, Global ischemia and Cardioplegia treated New Zealnad White Rabbit heart tissue using Trizol reagent (Gibco BRL- Life Technologies), according to the manufacturer’s protocol. Integrity of the RNA was determined by 1% agarose gel containing 3% formaldehyde, 0.02 M MOPS pH 7.4 (3-[N-morpholino] propanesulfonic acid) and 0.001 M disodium EDTA (ethylenediamine tetraacetic acid).RNA (35 ug) from control and experimental groups was used separately for first strand cDNA synthesis using an oligo-dT primer (0.5ug /uL). Following incubation at 65 oC for 5 min. the reaction was put on ice and the following added: 4uL first strand buffer; 2uL 0.1M DTT; 2uL 10X low dTTP–dNTP (5 mM dATP, dCTP, dGTP, and 2 mM dTTP); 2uL CyanineX-dUTP (1 mM ; Cyanine3; Cy3 for control; green and Cyanine5; Cy5 for experimental; Amersham, Pharmacia Biotech); 1uL RNasin and 1uL of Superscript II (GIBCO) and incubated at 42 oC for 30 min. then and another 1uL Superscript II added and the reaction allowed to continue a further 40 min. at 42 oC. The reaction was stopped with the addition of 2.5 uL 500 mM EDTA and heating to 65 oC for 1 min. The RNA template was hydrolyzed with the addition of 5 uL 1M NaOH and incubation at 65 oC for 10 min. and then neutralized by adding 12.5 uL 1M Tris pH 7.5 immediately to neutralize the pH. The labeled cDNA probes were purified by gel exclusion chromatography (ProbeQuant G-50, Amersham) and the volume reduced to 5-10 uL using a Speedvac.
Hybridization protocol
Contro heart samples were labeled with Cy3-dUTP and experimental hearts samples (Global ischemia and cardioplegia) were labeled with Cy5-dUTP and then combined with 30 uL hybridization solution (stock solution containing 100 uL DIG EasyHyb hybridization solution (Roche), 5 uL yeast tRNA (10 mg/mL), 5 uL human COT1 DNA, 5 uL Poly-dA, and 5 uL salmon sperm DNA (10 mg/mL). In the case of Global Ischemia vs. Cardioplegia, Global ischemia samples were labeled with Cy3-dUTP and Cardioplegia heart samples were labeled with Cy5-dUTP and then combined with 30 uL hybridization solution. The probe solution was hybridized to the arrayed slide at 37 C overnight. The following day, slides were washed first with 1x SSC to remove the cover slip (Hybri-Slips, Sigma) , then 3 successive washes of 1x SSC / 0.1% SDS for 15 minutes each at 50 oC, followed by a rinse with 1x SSC. The slides were dried in individual 50 mL conical tubes and centrifuged at 500 rpm to remove excess fluid.
Scan protocol
Scanning of the slides was performed using the GMS 418 scanner (Affymetrix) at 532 nm (Cy3) and 635 nm (Cy5).
Data processing
Local background was subtracted from the fluorescent value Cluster Analysis. All treatments were compared to Control to account for constitutive gene expression. Raw scanned images of Cy3 and Cy5 fluorescence were processed using Axon 4000A microarray scanner with GenePix analysis software using Ro etta Resolver (Molecular Devices Corp, Sunnyvale CA.). Dye swap was treated as technical replicates to account for dye effect error. Fold change criteria was used together with p-values to identify gene expression changes. p values were calculated based on dye swap results. Computational biology was performed using Spotfire Decision Site 8.1 (Spotfire, Somerville, MA). Functional annotation cluster analysis was performed using DAVID 2007 functional annotation bioiformatics microarray analysis (http://david.abcc.ncifcrf.gov/).
