4.1 Study approval
The protocol for the animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University with the reference number of IACUC2004003. The procedures were performed in accordance with the approved guidelines. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.
4.2 Animal preparation
Male Sprague-Dawley rats aged 14-16 weeks and weighing 350-450 g were housed in a pathogen-free environment with humidity between 45% and 55% and temperature maintained at 23-25°C. They were kept on a 12-hour light/dark cycle and provided with unlimited food and water for a week. All animals were purchased from the Animal Experimental Center of Nanjing Medical University (Jiangsu, China).
4.3 Anesthesia
The test subjects were first given sedatives and then kept under anesthesia using 2–2.5% isoflurane in a mixture of oxygen, with a FiO2 of 0.5. They were positioned dorsally with the inclusion of a rectal probe designed for thermocouple temperature measurements. To ensure thermal homeostasis, a controlled heating platform sustained their core temperatures between 37.5 and 38.5 Celsius. Venous access was established in the right femoral vein using a 24-gauge catheter (procured from BD Medical, Sandy, UT, USA) to enable the intravenous administration of pharmacological agents and fluid replenishment. At the same time, a catheter of the same size was placed in the right femoral artery to allow for careful monitoring of arterial pressure and to collect samples for blood gas analysis. The method of inserting a 14-gauge IV catheter into the trachea was completed, followed by connecting the subjects to a specialized rodent ventilator from Teli Anesthesia Breathing Equipment Company in Jiangxi, China. The ventilator settings included an FiO2 of 0.21, a tidal volume based on body weight (8-10 ml/kg), a PEEP of 5 cm H2O, and a respiratory rate set between 60 and 80 breaths per minute to ensure normal oxygen and carbon dioxide levels. Following this preparatory phase, a regime of complete intravenous anesthesia was instigated with an infusion of midazolam and fentanyl (administered at dosages of 7 mg/kg/h for midazolam and 70 μg/kg/h for fentanyl, sourced respectively from Hameln Pharma Plus, Germany and Rotexmedica, Germany), which consequently facilitated the discontinuation of isoflurane.
4.4 CPB circuit Preparation
The compact CPB system consisted of a venous reservoir, a sterile silicone tubing circuit with a 4 mm inner diameter for the venous line and 1.6 mm for the arterial line, a custom membranous oxygenator for small animals, and a small roller pump primed with 7 mL of saline and 1 mL (1000 IU) of heparin. The starting blend, made up of 6% HES 130/0.4, NaCl (Chiatai TianQing, Nanjing, China), 5% NaHCO3, and 20% human serum albumin (diluted to 5%), had a volume less than 8ml. (Figure 1)
4.5 Establishment of CPB
A 22-gauge catheter (BD Medical, Sandy, UT) was inserted into the caudal artery serving as the CPB inflow cannula. Following the insertion of the arterial inflow cannula, 500 IU/kg of heparin sodium was given. A modified 18-gauge catheter (BD Medical, Sandy, UT) with five side-holes was used to achieve CPB outflow, being inserted into the right atrium through the right jugular vein. A 20-gauge catheter, functioning as a tube for cardioplegia perfusion, was inserted in a retrograde manner into the ascending aorta through the right common carotid artery. (Figure 2)
4.6 The management of CPB
Following the insertion of arterial and venous tubes and connecting them, we turned on the oxygen before starting the CPB. The parameters were adjusted as follows: oxygen flow was 0.8 L/min, FiO2 100%, the respiratory rate was down to 30 per minute, the pressure of arterial oxygen was 200-400 mmHg. Prior to initiating CPB, the temperature setting for heating was raised to a maximum of 42℃ in order to prevent a sudden decrease in body temperature when CPB started. CPB was started with a flow rate of 100-150 ml/kg/min and rapid cooling to a rectal temperature of 32°C was promptly carried out. Moreover, the MAP was above 60 mmHg and the Hct was above 21% during the CPB.
4.7 The management of surgical procedure
Surgical procedure management included cardioplegic arrest and resuscitation following a right thoracotomy at the 4-5 intercostal space. Tourniquets were utilized to clamp the aortic arch and brachiocephalic trunk. Following 15 minutes of cardiopulmonary bypass, the aortic cross-clamp was placed as 0.5 milliliters of cardioplegia was quickly delivered through a cardioplegia perfusion tube using an infusion pump to induce diastolic cardiac arrest. If automatic heart resuscitated, 0.5 ml cardioplegic solution was administered again. Table 5 displayed the specific ingredients of the cardioplegia solutions. The specific infusion methods and dosages of the three groups of cardioplegia were shown in Table 6. Following 30 minutes of myocardial ischemia, the aortic cross-clamp was removed and the heart underwent reperfusion while being warmed to a rectal temperature of 36°C.At the same time, calcium chloride (Shen Wei Pharmaceutical Co.Ltd.10 mL/g 0.3mL, Sichuan, China) and epinephrine (Sanchine Pharmaceutical Co. Ltd. 1 mL/1 mg, Heilongjiang, China, 5ug) were given one after the other to help revive the cardiac muscle. Following a 15-minute reperfusion period, the animals were removed from cardiopulmonary bypass. Following CPB, the leftover priming solution was concentrated using the Heraeus 600i centrifuge to increase hematocrit levels before being injected into the rat. Subsequently, the cannulas were extracted, and the incisions were stitched. After monitoring post-CPB for a duration of 2 hours, all the animals were subsequently euthanized. The heart and blood were sampled after euthanized for further evaluation.
4.8 Specific Procedure for Carbon Dioxide Euthanasia
(1) Box packing: a. Initiate the process by uncovering the container housing the euthanasia apparatus; b. Position the cage box within the euthanasia box, with the density of the bottom side being at 3/4 capacity, and release the subject into the euthanasia box (ensuring the cage cover is removed while maintaining the retaining grid); c. Re-cover and securely seal the container. (2) Ventilation: a. Sequentially open the cylinder valve and decompression valve to facilitate ventilation for a period of 2 minutes; b. Upon observing clinical signs of death such as lack of breath or motion, continue perfusion for an additional 1 minute before closing the decompression valve and then the cylinder valve. (3) Standing: Confirm cessation of breathing and absence of heartbeat in the rats for a minimum duration of 5 minutes. (4) Placement of Deceased Animal in Enclosure: Transfer the deceased subject into a sealed bag, ensuring the body is first wrapped in paper and then placed into an opaque PE bag. (5) Sealing and storage: Seal the PE bag and store it in a refrigerator.
4.9 The standard for inducing cardiac arrest and reviving
(1) Continuous monitoring indicating a change in blood flow from variation to convection was the criteria for cardioplegic arrest. (2) Electrocardiogram readings indicating the absence of heart rate or a heart rate less than 20 beats per minute. (3) Femoral artery blood pressure dropping below 20 mmHg. Satisfying two out of the three conditions was seen as a sign of cardioplegic arrest(6).
Continuous monitoring of the femoral artery was necessary to achieve return of spontaneous circulation (ROSC), ensuring a steady and sustained pulsatile waveform while maintaining a pressure of 60 mmHg or higher. Furthermore, the rats showed a regular sinus rhythm on the electrocardiogram recordings.
4.10 Blood concentration
The blood was concentrated using a Heraeus 600i high-capacity low-temperature centrifuge during the process. The specified conditions were adjusted to 1000 rotations over a duration of 10 minutes. After spinning at 1000g for 1 minute, the liquid above the sediment was discarded, and the concentrated hemoglobin in the CPB system was injected into the caudal artery to achieve the target hematocrit level.
4.11 Outcomes
Animal survival was evaluated based on several factors. After CPB, the rats were observed for 2 hours. Vital signs like blood pressure, pulse, breathing rate, and peripheral oxygen levels were closely observed. Survival of the animals was determined by the stability and maintenance of these indicators within baseline values.
4.12 Blood gas and biochemical parameter analysis and histology
A femoral artery blood sample was utilized to evaluate the oxygen levels and metabolic condition (EG7+, iStar, Abbott Co. Ltd) at nine specific time intervals: before CPB (T1), at the start of CPB (T2), 10 minutes after CPB (T3), during cardioplegic arrest (T4), 10 minutes post-cardioplegic arrest (T5), 20 minutes post-cardioplegic arrest (T6), during cardiac resuscitation (T7), at the end of CPB (T8), and 2 hours after CPB (T9).The ventricular muscle tissues were taken after 2h observation. Partial myocardial tissues were collected and then immersed in 4% para-formaldehyde solution for 36 hours at a temperature of 4 degrees Celsius. Afterward, the tissues underwent dehydration with a LeicaCM1860 automatic dehydration machine from Germany, followed by embedding in paraffin and sectioning to a thickness of 4 μM. After that, the parts were dyed with Hematoxylin and eosin (HE) using an automated staining device (LeicaST5010, Germany). The other myocardial tissue was fixed in glutaraldehyde phosphate solution and placed in refrigerator at 4℃ for 1 week. After rinsed, fixed, dehydrated and soaked, each specimen was sliced with 2 copper mesh sheets. The ultrastructure of myocardial cells was observed under transmission electron microscope.
The standard of histopathological score:
The scoring system includes points for normal (1 point) or disordered (2 points) myocardial structure, absent (1 point), mild (2 points), or severe (3 points) myocardial edema, absent (1 point), mild (2 points), moderate (3 points), or severe (4 points) inflammation, and absent (1 point), scattered cells with ischemic necrosis present (2 points), visible aggregation of ischemic necrosis cells (3 points), or visible dissolved ischemic necrosis cells (4 points) for ischemic necrosis(7).
The standard of mitochondrial damage score:
Grade 0 indicated a typical mitochondrial structure with intact particles; Grade I showed a mostly normal structure with some particle loss; Grade II displayed swollen mitochondria with a clear matrix; Grade III exhibited fractured ridges and a solidified matrix; Grade IV demonstrated a total breakdown of membrane integrity, resulting in a vacuolar appearance(7).
4.13 ELISA arrays
Blood samples were taken from the femoral artery before and 2 hours after CPB. Levels of LDH, LDH-1, CK, and CKMB in the serum were analyzed with an ELISA Kit from Mlbio in Shanghai, China, according to the provided guidelines.
4.14 Western blot
Proteomic extraction was accomplished from the left ventricular myocardial tissue. The homogenization process utilized RIPA lysis buffer (supplied by Proteintech, Chicago, USA), augmented with both protease and phosphatase inhibitory substances (sourced from Biosharp, Beijing, China). Centrifugal separation ensued at 12,000 revolutions per minute for a duration of 15 minutes, after which the supernatant layers were sequestered. Protein measurement was conducted using the BCA Protein Assay Kit manufactured by Solarbio in Beijing, China. Protein concentrations were standardized across samples by carefully adding RIPA lysis buffer. Subsequent to this adjustment, protein fractions were segregated via electrophoretic migration on 10% SDS-polyacrylamide gels, followed by transference onto polyvinylidene fluoride membranes procured from Millipore, Massachusetts, USA. After that, the membranes were blocked using 5% non-fat milk from Nestle in Switzerland for 60 minutes at room temperature. Following this, the primary antibodies were incubated overnight at 4°C using Proteintech antibodies that were diluted. Following intermittent rinsing with Tris-buffered saline and Tween 20, membranes were introduced to horseradish peroxidase-linked secondary antibodies (also obtained from Proteintech, Chicago, USA) under room temperature conditions for a span of 1 hour. Visual detection of protein ligatures was facilitated by an amplified chemiluminescence detection reagent provided by Beyotime, Shanghai, China, and imaged with the Tanon 5200 Multi detection system (Shanghai, China). Image J software (Version 1.51, National Institutes of Health, USA) was used to perform quantitative band analysis.
4.15 Data analysis
The mean ± standard deviation was shown for all values. Statistical analysis was performed using GraphPad Prism 8 software by GraphPad Software Inc. Intergroup comparison was done through univariate analysis, and group comparison was conducted using the paired t test. A P-value less than 0.05 was deemed statistically significant.