Animals
The experiments were performed in adherence with the National Institutes of Health Guidelines on the use of Laboratory Animals and after approval of the protocol by the regional council of Tübingen (Registration number 905). Eight downsized familial hypercholesterolemia Bretoncelles Meishan (FBM) pigs of either sex with a mean bodyweight of 64 kg (range 24–92 kg) were used. The pigs were housed and fed as described previously [11, 12]. Na2S for i.v. injection was kindly provided by Csaba Szabo (Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA) and synthetized using H2S gas as the starting material, which was bubbled through an aqueous solution of sodium hydroxide (NaOH) and saline, formulated to pH neutrality and iso-osmolarity. This solution was filtered and placed under N2 atmosphere [13,14,15].
Procedure
The anesthetic procedure, surgical preparation, placement of catheters, and physiological measurements have been described in detail previously [12, 16]. Briefly, after induction of anesthesia (i.v., propofol, ketamine) and subsequent endotracheal intubation, anesthesia was maintained with continuous i.v. propofol (6–8 mg kg−1 h−1) and remifentanil (15–20 μg kg−1 h− 1). Pigs were mechanically ventilated [FiO2 0.25–0.35, adjusted to keep arterial pO2 levels > 100 mmHg, tidal volume 10 mL kg−1, positive end-expiratory pressure (PEEP) 5 cmH2O, inspiratory/expiratory time ratio 1:1.5, respiratory rate 10–13 min−1 adjusted to maintain arterial pCO2 between 35 and 40 mmHg]. These ventilator settings were used because swine are particularly susceptible to atelectasis formation in dependent lung regions due to the lack of alveolar collateral ventilation [16]. Sodium heparin (200 IU h−1) was continuously infused for anticoagulation. Via surgical cut-downs, the catheters were placed in the A. carotis dextra for measurement of blood pressure in the upper half of the body (mean arterial pressure, MAP proximal), trans-pulmonary single indicator thermodilution-cardiac index (CI), and the intra-thoracic blood volume index (ITBVI), a well-accepted marker of cardiac preload [17], as well as in the V. jugularis dextra for measurement of central venous pressure (CVP) and drug infusion. Via femoral cut-down, catheter sheaths were introduced into the Aa. femorales sinistra and dextra for distal blood pressure recording (MAP distal) and placement of inflatable balloon catheters. Adapting a technique previously published by several authors [18, 19], one catheter was placed directly above the aortic trifurcation, and the other one directly downstream of the A. subclavia sinistra, the correct position of which was manually controlled via a left-sided thoracotomy. This approach was chosen to prevent any perfusion of the spinal cord via collateral flow distal to the proximal balloon [20], which could result from variable bifurcation of the A. radicularis magna anterior [21]. The intra-aortic balloon occlusion was used to avoid mechanical injury related to clamp placement and release per se [22]. After 30 to 45 min of occlusion time, the balloon catheters were deflated. We started with an occlusion time of 30 min, followed by 40 min in preliminary experiments to test the response of this pig strain to a spinal cord I/R injury. Aortic occlusion was then performed for 45 min because this ischemia time had resulted in moderate neuronal damage (5 to 15% of all motor neurons) in the spinal cord in previous experiments [23]. In addition, 45 min of aortic occlusion prevented both the large spinal cord infarction over several segments reported in pigs after a clamping period of 60 min or longer [24] and the fairly mild tissue damage observed after only 30 min of clamping [25, 26]. Pigs were normothermic at the beginning of preparation period, and body temperature dropped to 32 °C at the start of either sodium sulfide or sodium chloride infusion. Pigs of this particular strain are known to spontaneously develop hypothermia during anesthesia [12]. Hemodynamic data, motor evoked potentials (MEPs), and neuronal function were assessed during aortic balloon occlusion and at 1 h, 4 h, and 8 h of reperfusion.
Application of sodium sulfide (Na2S)
Two hours before balloon occlusion, animals were treated with saline (vehicle group) or sodium sulfide (study group). The sodium sulfide infusion rate (initial bolus 0.2 mg kg−1 followed by continuous i.v. infusion of 2 mg kg−1 h−1 during the 2 h before aortic occlusion, and 1 mg kg−1 h−1 during the 8 h reperfusion) was based on previous studies [27]. During balloon occlusion time, sodium sulfide or vehicle infusion was stopped.
Harvesting, measurements, and calculations
The animals were euthanized in deep anesthesia by giving a bolus of phenobarbital (Narkodorm Alvetra, Neumünster, Germany) and i.v. potassium chloride (20 mmol, Braun, Melsungen, Germany). Tissue samples of the lumbar and thoracic spinal cord were isolated immediately post mortem and fixed in 6% buffered paraformaldehyde, and standard 3-μm paraffin sections were stained with hematoxylin and eosin (HE). Particular attention was paid on the anterior horn (AH), because of the vulnerability due to a hypoxic damage [28]. In addition, spinal cord sections were analyzed after nuclear cresol violet staining (Nissl staining) for neuronal damage [26].
Spinal cord function was evaluated by MEPs as described previously [24, 29, 29, 30]. Three electrodes were inserted into the scalp, and one into the soft palate to apply electric impulses (Digitimer Ltd., MultiPulse Stimulator D185 mark IIa) to the motor cortex. To quantify MEPs, electrodes were inserted in the muscles of the limbs to measure neuronal potential (ExcelTech Ltd., ExlTek Neuromax 1004). After electric stimulation of the cerebral motor cortex, the neural responses of the upper and the lower limbs were recorded. Decrease of more than 75% of the MEP amplitude was accepted as an indication of ischemic spinal cord dysfunction [24, 29]. MEP signal disappeared within 5 min in all animals as a sign of sufficient aortic occlusion. MEPs were triggered directly before aortic occlusion as well as 1 h, 2 h, 4 h, and 8 h after reperfusion.
In addition, spinal cord function was clinically evaluated by observing the movements of the upper and lower limbs in response to claw clamping during temporarily reduced anesthesia. The muscular response was classified as follows: 0 = no movement, 1 = muscular movement, 2 = joint movement, 3 = normal movement; an additional score of 4 was attributed if spontaneous movement was present even without stimulation by claw clamping. The reaction was measured before clamping (measuring point, MP1) and 1 h (MP2), 2 h (MP3), 4 h (MP4), and 8 h (MP5) after reperfusion. The reaction of the upper extremities was used to demonstrate the normal response.
Heart rate, MAP proximal, MAP distal, CVP, ITBVI, and CI were recorded as hemodynamic parameters. Arterial blood samples were collected to analyze blood gases, acid-base status, electrolytes, hemoglobin content, O2 saturation, and glucose levels.
Statistical analysis
All data are presented as median (interquartile range, IQR). A Mann-Whitney U test was performed for systemic hemodynamics, gas exchange, acid-base status, and histology. A p value of less than 0.05 was considered statistically significant. IBM SPSS Statistics software (Version 24.0.0.0) was used for statistical evaluation and graphical display.