This study was ethically approved (2018/1375) by the University of Sydney Animal Ethics Committee and conducted in accordance to the University of Sydney Animal Ethics Committee guideline. The Institutional Animal ethics committee complies with the Australian NHMRC rules for animal experimentation and international regulations including the Declaration of Helsinki. The study is reported in accordance with ARRIVE guidelines.
Animals
Eight healthy male white sus domesticus pigs were obtained from a commercial piggery and acclimatised in the facility for at least 7 days prior to any experiments. Solid pig weaning feed and water were made available ad libitum for the entire duration of the animals’ stay. Animals’ mean weights were weighing 52 ± 6 kg.
Study design
Animals (n = 8) were randomly assigned to a protocol in the 8-day study. Animals were deemed healthy on pre-anaesthetic physical examination. Each animal was anaesthetised and catheterised for pressure and flow measurements. They were then stimulated as described in the stimulation methods section and pressure/hemodynamic recordings were recorded (Blood flow and pressure measurements section) before and post-stimulation at multiple arterial locations (renal, hepatic, coronary, and carotid arteries). The animals were euthanized humanely with intravenous barbiturates at the end of each day.
Experimental protocol
Animals were sedated with tiletamine/zolazepam 6mg/kg (Zoletil®, Virbac, Australia) and dexmedetomidine 12 mcg/kg (Dexdomitor®, Zoetis, Australia). Anaesthesia was induced with 4–6mg/kg of propofol (Propofol-Lipuro 1%®, B. Braun, Germany) intravenously. Anaesthesia was maintained with propofol 8 mg/kg/hr, ketamine 5 mg/kg/hr (Ketamil®, Troy Laboratories, Australia) and dexmedetomidine 2-4mcg/kg/hr administered intravenously via individual syringe drivers. This TIVA protocol was previously demonstrated to have negligible effects on vascular tone and blood pressure [14].
Vital signs (pulse oximetry, invasive blood pressure, heart rate, electrocardiogram, end tidal carbon dioxide, and oesophageal temperature) were continuously monitored throughout the procedure (IntelliVue Mx800 system, Philips Healthcare, Australia). Arterial blood gas analysis was performed intermittently throughout anaesthesia to assess physiological status and ensure normal organ perfusion and ventilation.
Stimulation
Non-invasive transcutaneous electrostimulation electrodes (TENS) were used to facilitate the electrical stimulation. A TENS stimulator (SBEX, Texas Southwest Research Institute, Texas, US) delivered stimulation in electric pulses with a current of 35 mA and a pulse width of 180 ms. Stimulations were administered for 10 minutes each at three different frequencies: high frequency of 80Hz, a low frequency of 10 Hz, and a placebo stimulation with 0 Hz. At least 20-min was allowed in between each stimulation to allow the return to baseline blood flow levels. Two types of electrode configurations, abdominal only and abdominal leg stimulation orientations (Fig. 1A). The stimulation modalities/parameters were single-blinded for the research team (imaging team were blinded for the stimulation modalities/parameters).
Abdominal only configuration of the electrodes
For the abdominal only (Fig. 1A, left) configuration, 4 TENS electrodes were placed 2 cm inferiorly and superior to the umbilicus and 3 cm apart laterally from the midline passing through the umbilicus on each side to form a rectangle shape configuration.
Abdominal and Hind Limb combined configuration of the electrodes
For the abdominal and hind limb (Fig. 1A, Right) stimulation two electrodes were placed 2cm inferior to the umbilicus and 3 cm lateral to midline (same as the abdominal only orientation) while the other 2 electrodes were placed on the hind limbs.
Each electrode configuration and stimulation frequency were randomised across the animals to reduce confounding effects. The randomisation of the stimulations over each pig are presented in Table 1.
Table 1
Randomisation of stimulation protocols. A pig for each day (in total 8 pigs) is used for the study. (Configuration of electrodes, AL: Abdomen and Hind Limb, A: Abdomen only)
Pig
|
Protocol 1
|
Protocol 2
|
Protocol 1
|
|
|
Protocol 2
|
|
|
1
|
AL
|
A
|
Placebo
|
10 Hz
|
80 Hz
|
80 Hz
|
Placebo
|
10 Hz
|
2
|
AL
|
A
|
80 Hz
|
10 Hz
|
Placebo
|
Placebo
|
10 Hz
|
80 Hz
|
3
|
A
|
AL
|
Placebo
|
80 Hz
|
10 Hz
|
80 Hz
|
10 Hz
|
Placebo
|
4
|
AL
|
A
|
80 Hz
|
Placebo
|
10 Hz
|
10 Hz
|
80 Hz
|
Placebo
|
5
|
A
|
AL
|
10 Hz
|
Placebo
|
80 Hz
|
10 Hz
|
Placebo
|
80 Hz
|
6
|
A
|
AL
|
10 Hz
|
80 Hz
|
Placebo
|
80 Hz
|
10 Hz
|
Placebo
|
7
|
AL
|
A
|
80 Hz
|
Placebo
|
10 Hz
|
Placebo
|
80 Hz
|
10 Hz
|
8
|
A
|
AL
|
10 Hz
|
80 Hz
|
Placebo
|
Placebo
|
10 Hz
|
80 Hz
|
Blood flow and pressure measurements
To measure the pressure and haemodynamic responses due to stimulation, the animals were catheterised with flow and pressure sensors. Two 6F sheaths were percutaneously placed in each femoral artery. One additional 7F sheath was placed in the left femoral artery for blood pressure measurement. Four 6F Cordis Hockey Sticks guide catheters were inserted through a 6F sheath for each of the following arteries (Fig. 1B), left renal, common hepatic, anterior interventricular artery, and left carotid arteries. A mixture of equal parts saline and iodine-based non-ionic contrast agent, iohexol, 647 mg/ml, equivalent to 300mg iodine per ml (Omnipaque-300, GE Healthcare, Chicago, IL) was used via hand injection to visualize the targeted arteries for any vessel spasm and dissection.
Two minutes before and following stimulation, blood flow and pressure measurements were taken. Vessels of multiple organs were chosen to investigate the effects of stimulation, these include: the left renal artery to assess renal blood flow, common hepatic artery for hepatic flow via a branch of coeliac trunk, and the left coronary artery for coronary blood flow. Care was taken to keep the wire tip at the centre of the artery lumen for optimal measurements. The positioning of the catheters for each artery was kept consistent in between sessions with the aid of the previous session’s x-ray capture.
The pressure and flow velocity signals, were measured simultaneously using the Combowire® (Volcano Corporation, San Diego, CA, USA) and data recording software (Labchart, ADInstruments, Bella Vista, NSW Australia) via data acquisition hardware device (Powerlab 16/35 box, ADInstruments, Bella Vista, NSW, Australia). Simultaneous recordings of aortic/arterial pressure (via femoral artery) and ECG signals were also acquired (Phillips IntelliVue MX800, Cambridge MA, USA).
Cerebral measurements
For cerebral (intracranial) and extracranial arterial blood flow, digital subtraction angiography (DSA) protocols were applied. The common carotid artery was injected via the catheter with 10 ml of the iodine-based non-ionic contrast agent, iohexol, 647 mg/ml, equivalent to 300mg iodine per ml (Omnipaque-300, GE Healthcare, Chicago, IL) using a power-contrast injector (Angiomat Illumina, Liebel-Flarsheim, USA) over 2 s (flow rate 5mls/sec). The tip of the catheter was kept in the common carotid artery before and after the stimulation. The positioning of the catheters for each artery was kept consistent in between sessions with the aid of the previous session’s x-ray capture. Angiographic images were acquired at a rate of 7.5 fps in anterior-posterior, lateral position, with a robotic c-arm system (Artis Pheno, Siemens Healthineers, Erlangen, Germany). Post-processing analyses of cerebral blood flow was conducted with Syngo iFlow (Siemens Healthineers, Erlangen, Germany).
The order of flow (and pressure) recordings of each organ for each session were as follows. The baseline recordings followed the order of renal, common hepatic, coronary arteries and cerebral circulation and post-stimulation recordings were obtained in the order of cerebral, coronary, common hepatic and renal arteries (Fig. 1B Left and Fig. 2).
In between each organ recording, there was 1 minute of catheter travel time and 2 minutes of recording time for each organ’s artery (Fig. 2).
Data analysis
Data sets were stored digitally and analysed offline using MATLAB (R2019b, Mathworks, MA, USA) with a custom-made software package.
Analysis of pressure-flow velocity-derived indices in intraarterial Catheter Recordings
Average peak flow velocity (APV) in cm/s, distal pressure taken by the pressure catheters (PD) and aortic-arterial pressure (PA) taken by a blood pressure machine at the renal, celiac and coronary arteries were investigated to determine stimulation-based modulation pressure/flow. Data for each experimental condition was averaged over 15 seconds (30 seconds into the session) during the recordings for each organ location. All analyses were performed in a fully automated fashion, removing the need for manual selection of data time points.
Intracranial and extracranial blood flow Analysis with DSA-Syngo iflow
Regions of interests (ROIs) were selected distally to the common carotid artery (CCA), ROI1 on maxillary artery, a branch of external carotid artery for the extracranial artery and the ascending pharyngeal artery (ROI2) and internal carotid artery (ICA) (ROI3) was selected for intracranial arterial blood flow analyses in Syngo iflow-color- coded DSA (Fig. 3). Each of the ROIs peak which provide indication blood flow metrics were determined using the commercially available post-processing software Syngo iFlow (Siemens Healthineers, Erlangen, Germany). For each ROI, a time-versus-intensity graph was exported by the software with calculated metrics of: 1) ROI peak time: time for the contrast intensity of a ROI to reach peak value; 2) ROI arrival time: the time of arrival of contrast material; 3) MTT: mean transit time, i.e. average contrast transit time through the ROI, measured as the time difference between each of the arterial ROI peaks; and 4) TTP: time to peak i.e. time from the first appearance of contrast in the ROI to the peak contrast concentration in the ROI.
Statistical analyses
Statistical analyses were performed in one-way ANOVA to investigate the effects of placebo, 10 Hz and 80 Hz stimulations on the characteristics of blood flow, i.e., velocity, distal pressure, and arterial pressure, quantified by the mean, standard error of the mean (SEM) and p-value. A p-value of less than 0.05 was considered to be statistically significant. A Tukey Kramer follow-up test was used to quantify the differences where statistical significance was found.