Seven ovine eyes from animals of similar age, between 1 and 2 years old, were obtained from a local abattoir. Eyes were cleaned, rinsed and stored in PBS at 4°C prior to testing. All eyes were processed and imaged within 48 hours of death. Eyes were cannulated through the anterior chamber to allow for control of IOP inside the MRI scanner. IOP was varied by changing the height of PBS in a gravity perfusion system as previously described (Ho et al., 2016 (link); Ho et al., 2014b (link)) and monitored by a pressure transducer designed for MRI applications with a sensitivity of 1 mmHg (BIOPAC Systems, Goleta, CA, USA). Eyes were suspended in hydrogel in an open container, as we have described previously, to prevent the application of external forces (Ho et al., 2016 (link)). The same eyes were scanned serially under increasing levels of IOP: 0 mmHg, 10 mmHg, 20 mmHg, or 40 mmHg. Eyes were allowed to equilibrate for 20 minutes after an IOP increase before scanning. An equilibration time of 20 minutes was chosen as we have previously demonstrated that only minimal tissue deformation occurs after 15 minutes of equilibration in topographic images of the vitreo-retinal interface (Sigal et al., 2005 (link)). The pressure transducer was used to verify that a constant pressure was applied at the desired level during each experimental session.
Pressure transducer
Manufactured by Biopac
Sourced in United States
A pressure transducer is a device that converts pressure into an electrical signal. It measures the pressure of liquids or gases and generates an output voltage or current that is proportional to the applied pressure.
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9 protocols using pressure transducer
1
Ovine Eye MRI at Varying IOP
2
Imaging Corneal-Scleral Biomechanics via MRI
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Twelve fresh, unfixed ovine eyes underwent anterior chamber perfusion in the 9.4 Tesla MRI scanner using a plastic cannula connected to a saline bag (0.9% sodium chloride; Baxter International Inc., Deerfield, IL, USA) and a pressure transducer (BIOPAC Systems, Goleta, CA, USA) so as to image the dynamic effects of stepwise changes in IOP on the tissue transverse relaxation times (T2) of the corneoscleral shell in the same eyes (Fig. 1a,b ). Six of these 12 ovine eyes were loaded at 0, 10, 20 and 40 mmHg by raising the saline bag at different heights consecutively inside the MRI scanner, followed by unpressurization back to 0 mmHg until the end of the experiment. Although 0 mmHg is not a normal physiological condition, it was selected as the lowest pressure to provide fundamental insights into the state of the tissues without the load from IOP, which is essential in the formulation of a comprehensive mechanistic model of the tissues5 (link)53 (link)54 74 (link). The pressure transducer was used to ensure a constant pressure was applied at the desired level during each experimental session. Six other fresh ovine eyes were cannulated but kept at 0 mmHg throughout the entire MRI experiment as an unloaded control. Hydrogel (McNeil-PPC, Inc., Skillman, NJ, USA) was applied to keep the surface of the ovine eyes moist throughout the experiment.
3
High-Resolution Structural Brain Imaging
4
Evaluation of Penile Erectile Function
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After the 12-week treatment, erectile function was assessed using intracavernosal pressure (ICP) and mean arterial pressure (MAP) tests as previously reported [12 (link)]. Briefly, while under anesthesia, the bilateral cavernous nerves were exposed and a bipolar steel electrode was used for nerve stimulation. The corpora at the middle of penile shaft were cannulated with a 25-G needle containing heparin (100 U/ml) connected to a pressure transducer (Biopac Systems Inc., Goleta, California, USA) to record ICP. Stimulation parameters were 5 mA, 20 Hz, plus 0.2 ms, and duration of 60 s. The mean MAP was calculated after electrical stimulation. The erectile evaluation was measured as mean ICP, MAP, and the ratio of ICP and MAP. The mean ICP and MAP were recorded using AcqKnowledge®4.4 software (Biopac Systems Inc. Goleta, California, USA). After data recording, the blood samples and the shaft of the penis were harvested for analysis.
5
Invasive Pulmonary Arterial Blood Pressure Measurement
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Invasive pulmonary arterial blood pressure (PABPINV) measurements were performed at the 7th week. Before the procedure, rats were intubated with a 16-gauge plastic venflon, which was inserted directly into the trachea. Animals were subsequently attached to a mechanical ventilator (Harvard Apparatus 55-7059 inspira ASV ventilator, Holliston, MA, USA), ensuring normal breathing. Rats were ventilated under a peak pressure of 10 ± 2 cmH2O, a respiratory frequency of 60 ± 5 breath/min, and a FiO2 (fraction of inspired oxygen) value of 100%. After this, the mediastinum was opened by median sternotomy to locate the pulmonary artery. PABPINV was measured by a 26-gauge plastic venflon that was directly connected to a pressure transducer (Biopac System Inc.) and signals were continuously recorded.
6
Measuring Cavernous Nerve Stimulation and Intracavernosal Pressure
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To measure ICP with cavernous nerve stimulation, animals were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. The right carotid artery was cannulated with polyethylene tubing containing heparinized saline for continuous monitoring of mean arterial pressure (MAP). The shaft of the penis was then exposed from skin and muscle and punctured with a 23-gauge needle connected to the tubing. Both were connected to a pressure transducer (Biopac system Inc, Goleta, CA, USA). The cavernous nerve was stimulated using a bipolar electrode connected to a stimulator. Stimulator settings were 5 V of 1.5 mA for 1 min with a minimum interval between stimulation of 5 min. The ICP/MAP ratio was determined using the maximum ICP divided by the MAP obtained during cavernous nerve stimulation. All data were recorded by an MP100 data acquisition system and analyzed using Acqknowledge software (Biopac System Inc., Goleta, CA, USA).
7
Chronic Blood Pressure Monitoring in Late-Pregnant and RUPP+HC Rats
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Separate groups of normal late-pregnant (Late-Preg; n = 6) and RUPP+HC rats (n = 6) were implanted with indwelling carotid catheters on day 18 of gestation. Under isoflurane anesthesia, the left common carotid artery was exposed and cannulated with saline filled V-3 tubing (SCI) which was tunneled to the back of the neck and externalized. On day 19 of pregnancy, rats were lightly anesthetized with isoflurane (1–2% for 3–4 minutes) and a pressure transducer (BIOPAC, Inc., Goleta, CA, USA) connected to the indwelling carotid catheter. Rats were placed in a rectangular, clear plastic modular chamber (10”×7”×5”) with a metal grid floor, large enough to move freely. Average conscious, unrestrained systolic blood pressures were recorded using AcqKnowledge software (BIOPAC, Inc., Goleta, CA, USA). One RUPP+HC rat died due to surgical complications during catheter implantation and was excluded.
8
Cystometric Evaluation in Awake and Anesthetized Rats
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After recovering for 24 h, a CMG on fully awake and freely moving rats was conducted in a metabolic cage. The catheter was connected via a three‐way connector to an infusion pump for saline infusion (Braintree Scientific, Braintree, MA, USA), and a pressure transducer (Biopac Systems Inc., Santa Barbara, CA) for bladder pressure recording. Voiding responses were elicited by continuously infusing saline (0.9% NaCl) at a rate of 0.12 ml/min at room temperature (approximately 22°C). The same solution, temperature, and infusion rate were used in all three experiments. Urine was collected in a container coupled to a weight transducer (Biopac Systems Inc.). The cystometric evaluation was performed until 10 micturition cycles were recorded. After an additional 24 h, the same rats were anesthetized with 1.2 g/kg s.c. of urethane and a CMG test was once again performed. The CMG parameters analyzed included inter‐contraction interval, baseline bladder pressure, maximal bladder pressure, and voided volume.
9
Elevated Intraocular Pressure in Rats
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IOP was elevated in the right eye of forty-five Sprague-Dawley rats as described63 (link). Briefly, ketamine: xylazine anaethesia (75:10 mg/kg) was administered intraperitoneally and eyes received one drop each of proparacaine and tropicamide to induce analgesia and pupil dilation, respectively. A 30 g needle, connected to a saline reservoir (0.9% sodium chloride; Baxter International Inc., Deerfield, IL), was inserted into the anterior chamber parallel to the iris and secured in place. The reservoir was elevated to increase the IOP to 130 mmHg for 60 min. IOP was measured using a pressure transducer (BIOPAC Systems, Goleta, CA, USA) and a handheld tonometer (Icare TONOLAB, Finland). After 60 min, the reservoir was lowered, the needle removed, and a drop of gentamicin applied (Akorn, Lake Forest, IL).
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