Model of Ischemia and Reperfusion Injury in Rabbits

Summary

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

Abstract

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer's solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis.

With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located.

A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse.

For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively.

For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

Introduction

Cardiovascular diseases are the leading cause of death in the world and contribute to over 18 million deaths each year^1,2,3. Acute myocardial infarction (MI) is a common medical emergency that develops when a blood clot or a piece of atheromatous plaque blocks the blood flow of a coronary artery. This causes regional myocardial ischemia in the territory that the artery perfuses.

The present study describes a protocol that utilizes a simple and reliable methodology to create in situ acute regional myocardial ischemia in a rabbit model for non-survival and survival experiments. The initial goal of this method was to evaluate the effects of mitochondrial transplantation on modulating myocardial necrosis and increasing the post-ischemic heart function following an ischemic event. Previous research has demonstrated the occurrence of mitochondrial alterations and a rapid decline in high-energy phosphate levels following the onset of ischemia and a reduction in the oxygen supply, resulting in a drastic decrease in the cardiac energy stores⁴. Investigators have attempted to improve post-ischemic function and lessen myocardial tissue necrosis using pharmacological interventions and/or procedural techniques, but these techniques provide limited cardioprotection and have minimal impact on mitochondrial damage and dysfunction^5,6,7. Our team and others have previously shown that mitochondrial damage primarily occurs during ischemia and that contractile recovery can be enhanced and the myocardial infarct size decreased with the preservation of mitochondrial respiratory function during reperfusion^8,9,10. Thus, we hypothesized that mitochondrial transplantation from tissues unaffected by ischemia to the area of ischemia prior to reperfusion would provide an alternative approach to reduce myocardial necrosis and enhance myocardial function. Herein, we detail the protocol used to test this theory and the representative results obtained from our initial study analysis.

Furthermore, several investigators have focused on other topics integral to defining the impact of myocardial ischemia-reperfusion injury and establishing appropriate therapeutic interventions. One such area of research is that of preconditioning. Myocardial ischemic preconditioning is a cardioprotective mechanism activated by brief ischemic stress that results in a reduction in the rate of cardiac cell necrosis during subsequent episodes of prolonged ischemia. These mechanisms can be activated by either hypoxia or coronary occlusion. Mandel et al. demonstrated that hypoxic-hyperoxic preconditioning helped to maintain the balance of nitric oxide metabolites, reduced endothelin-1 hyperproduction, and supported organ protection¹¹. Moreover, the concept of remote ischemic preconditioning, a phenomenon whereby single-organ preconditioning provides systemic protection, has been explored. Ali et al. found that, in patients undergoing elective open abdominal aortic aneurysm repair, remote preconditioning, performed by intermittently cross-clamping the common iliac artery to serve as a stimulus, reduced the incidence of postoperative myocardial injury, myocardial infarction, and renal impairment¹².

Rabbit models offer potential advantages over models with other species and have been used in multiple different scenarios for decades, including the induction of arrhythmias, global and regional ischemic models, and cardiac contraction research, amongst others^13,14,15. Although the rabbit heart is smaller than that of a dog or pig, it is large enough to easily perform surgical procedures at a much lower cost¹³. The rabbit heart is often used as it closely parallels the human heart; indeed, it has a similar metabolic rate, expresses β-myosin heavy chain, andlacks significant myocardial xanthine oxidase¹⁶. The technique herein described to induce regional myocardial ischemia is simple, repeatable, and cost-effective. This method allows for both non-survival and survival cases, as only regional ischemia is induced rather than global ischemia, and the materials needed are non-specialized. Two different surgical approaches (i.e., sternotomy and mini-thoracotomy) can be utilized, thus providing the operator and experimental protocols more freedom in terms of the study design. Additionally, the procedure does not require the use of a cardiopulmonary bypass. In this context, minimally invasive approaches to coronary artery bypass grafting have become valuable alternatives for patients in need of multi-vessel revascularizaiton^17,18. This model could be used to study the differences between these approaches and provide an animal-based learning tool for surgical trainees. Additionally, performing cardiac catheterization utilizing this model may be useful for physiological research and/or surgical training.

Our model provides a methodology for applications in which inducing regional myocardial ischemia and subsequently measuring the infarct size, myocardial function, and cellular changes are of importance. With this protocol, we have been able to evaluate several markers of cellular function and adaptation to ischemia and the proposed therapeutic intervention (i.e., mitochondrial transplantation) by examining the internalization of organelles, oxygen consumption, high-energy phosphate synthesis, and the induction of cytokine mediators and proteomic pathways. These outcomes are important in preserving the myocardial energetics, cell viability, and cardiac function and allow for the objective evaluation of cardioprotective techniques following ischemia-reperfusion injury. This model could be used to study similar biologic pathways and alternatives in the field of post-ischemic myocardial pathology and recovery.

The goal of this protocol is to provide a highly reproducible methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. This model provides a methodology with high survival, low intraoperative mortality, and minimal morbidity¹⁹. Other models for acute regional myocardial ischemia have been described using radiolabeled materials, contrast agents, magnetic resonance imaging, or computer simulations^20,21,22. Our protocol provides a reliable and simple methodology that is cost-effective, consistently reproducible, and has a low technical demand and, thus, can be performed by investigators without surgical expertise. This protocol accommodates either a survival project using a left mini-thoracotomy or a non-survival model using a midline sternotomy.

Protocol

This investigation was conducted according to the National Institutes of Health's guidelines on animal care and use and was approved by the Boston Children's Hospital's Animal Care and Use Committee (Protocol 20-08-4247R). All the animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.

1. Animal species, anesthetic, and analgesic agents

1. Animal species: Use New Zealand White rabbits (wild type strain; female sex; sexually mature 15-20 weeks old; 3-4 kg body weight) for experimental studies.
2. Anesthetic and analgesic agents:
   1. Use atropine at a dose of 0.01 mg/kg intramuscular (IM)
   2. Use acepromazine at a dose of 0.5 mg/kg IM for initial sedation and 0.5 mg/kg intravenous (IV) for full anesthetization.
   3. Use butorphanol at a dose of 0.5 mg/kg IM.
   4. Use isoflurane via a precision vaporized system face mask at 3% for induction, followed by intubation at 1%-2%, oxygen (O2) at 100% at 2 L/min, and general anesthesia at 1% for maintenance.
   5. Use medetomidine at a dose of 0.25 mg/kg IM.
   6. Use ketamine at a dose of 10 mg/kg IV.
   7. Use a bupivacaine intercostal block at the thoracotomy site at a dose not exceeding 3 mg/kg IM.
   8. Use 1% lidocaine at a dose of 1-1.5 mL/kg IV.
   9. Use a 1-4 µg/kg fentanyl transdermal patch for 72 h.

2. Procedural steps (Figure 1)

1. Sedate New Zealand White adult rabbits with a single combined IM injection of atropine, acepromazine, and butorphanol. Induce the animal with 3% isoflurane via a precision vaporized system face mask.
2. Preparation prior to blinded endotracheal intubation (i.e., without visualization of the glottis)
   1. Spray the larynx with 1% lidocaine to prevent laryngospasm.
   2. Pre-measure the endotracheal tube (ETT) length on the outside of the rabbit from the teeth to the predicted carina, and place the rabbit in a sternal recumbency position with the neck extended.
3. Intubate the animal with a cuffed pediatric size (3-0 or 3-5 inner diameter) ETT under continuous inhaled anesthetic at 1%-2% and O₂ at 100% at 2 L/min.
   1. Insert the ETT into the mouth, and direct it past the torus into the pharynx.
   2. Advance the ETT until either the tip of the tube contacts the glottis or the breath sounds are lost, indicating the tube tip has passed through the glottic opening.
   3. Slightly withdraw the tube until the breath sounds are regained, and then re-advance again, and secure the tube in place.
4. Ventilate the animal with mechanical support (tidal volume: 10 mL/kg, fraction of inspired O₂: 40%, respiratory rate: 30-40 breaths/min, positive end-expiratory pressure: 5-10 cmH₂O).
   1. Adjust the FiO2 as tolerated to achieve an O₂ saturation greater than 92% as measured by pulse oximetry to prevent hyperoxia, which can provoke a systemic inflammatory response.
5. Verify the proper placement of the ETT by a physical exam (i.e., auscultation), clinical signs (i.e., observation of condensation at the end of the endotracheal tube), and with objective measures (i.e., end-tidal carbon dioxide).
6. After approximately 10 min, deliver an IM injection of medetomidine to the rabbit to provide simultaneous anesthetic and analgesic effects.
7. Maintain the general anesthesia with 1% isoflurane for the duration of the surgical procedure.
8. Insert a 22 G IV catheter into the marginal ear vein, and secure it with tape to obtain peripheral IV access.
   NOTE: The femoral vein can be used as an alternative site of venous access.
   1. Fully anesthetize the animal with acepromazine IV and ketamine IV.
   2. Prior to incision, inject 1,000 U/mL heparin at a dose of 3 mg/kg IV.
      1. Administer 1,000 U/mL heparin at a dose of 3 mg/kg initially, and re-dose hourly until the end of the experiment to maintain an activated clotting time of >400 s, in keeping with the current surgical protocol.
   3. Administer 1% lidocaine IV and/or epicardial asynchronized defibrillation as needed if ventricular fibrillation occurs during the surgery. Ventricular fibrillation usually stops with one or two doses of lidocaine.
   4. Perfuse lactate Ringer's solution continuously at 10 mL/kg/h.
      NOTE: Given the small volume of fluids administered and the short operative times, the animals in survival studies in this work did not require diuresis prior to extubation or during the recovery period. If the animal develops a worsening pulmonary status (i.e., increasing the ventilator settings, evidence of pulmonary edema on auscultation, etc.), diuresis is advised.
9. Perform a carotid cut down, and place a 4 or 5 French arterial line to facilitate the intra-operative monitoring of the arterial blood pressure (BP).
   NOTE: The femoral artery can be used as an alternative site of arterial access.
10. Monitor and record all the physiologic and mechanical variables by continuous real-time analysis.
    1. Monitor the arterial BP with the carotid arterial line, and record the O₂ saturation using pulse oximetry via a sensor placed on a shaved paw.
    2. Monitor with an electrocardiogram (ECG) with three limb leads: I, II, and III, and three computed augmented leads: aVL, aVR, and aVF.
       1. Record the ECG tracings at the pre-ischemic baseline, during ischemia, during reperfusion, and serially during days 7-28 of recovery (if performing a survival study).
    3. Monitor the level of sedation by continuous monitoring of the BP and heart rate (HR).
    4. Monitor the temperature with a rectal probe.
    5. Use 2D ECHO from the left parasternal and apical views to assess the myocardial function at desired time points in both the survival and non-survival cases.
       1. Assess the myocardial function using the fractional shortening (FS) by measuring the left ventricle end-diastolic distance (LVEDD) and the left ventricle end-systolic distance (LVESD) and using the following formula:
          FS = (LVEDD − LVESD)/LVEDD × 100
11. During surgery, place the animal on a circulating hot water blanket to maintain a stable core body temperature.
12. Prepare and drape the animal in a sterile fashion:
    1. Shave the surgical site, and prep with betadine and 70% isopropyl alcohol, each applied in triplicate. Pat the area dry with sterile gauze pads, and drape the entire animal with sterile towels.
13. Left mini-thoracotomy (survival studies)
    1. Perform an intercostal block at the predetermined thoracotomy site with bupivacaine IM.
    2. Administer 1% lidocaine IV via the auricular vein prior to incision.
    3. Perform a left mini-thoracotomy through the fourth intercostal space along the upper portion of the fifth rib to avoid the neuromuscular bundle, which is located parallel to the undersurface of each rib.
       1. Perform an anterolateral thoracotomy for the best visualization of the anterolateral surface of the heart (i.e., the anatomic location of the LAD diagonal branches).
       2. Position the rabbit with the left side elevated approximately 30° using a pillow or bean bag.
       3. Secure the rabbit's ipsilateral leg above its head to create room for both the operative field and between the rib spaces.
       4. Palpate and outline the bony landmarks, including the ribs, sternum, and scapula, with a felt-tipped marking pen. Incise the skin overlying the fifth rib using a #10 blade. Ensure that the incision remains parallel to the rib.
       5. Use electrocautery to divide the pectoralis major muscle and serratus anterior muscle. Divide the intercoastal muscles right above the fifth rib with electrocautery to preserve the neurovascular bundle.
       6. Cautiously enter the pleural space through the fourth intercostal space with sharp or blunt dissection. Extend the initial pleural incision parallel to the rib in both directions with sharp or blunt dissection until a rib spreader or sternal retractor can be inserted.
    4. Place a rib spreader or sternal retractor within the rib space, and widen to provide adequate visualization of the heart and pericardial sac. Lift the pericardium with DeBakey forceps, and open the pericardium with Metzenbaum scissors.
    5. LAD artery isolation
       1. Encircle the second or third diagonal branch of the LAD artery with a polypropylene suture (3-0) on a taper needle. Remove the needle, and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.
       2. Place a pledget between the snare and the coronary artery to avoid damaging the coronary and/or causing vasospasm with ligation.
          1. Using DeBakey forceps, pick up a rectangular PTFE felt pledget (approximately 7 mm x 3 mm). Place the pledget between the two polypropylene filaments so that it is sandwiched between the isolated LAD artery and the vinyl tube when the snare is tightened.
14. Midline sternotomy (non-survival studies)
    NOTE: The midline sternotomy approach is ideal for non-survival cases, for which more invasive monitoring with LVDP and sonomicrometry can be utilized.
    1. Perform a midline sternotomy using curved Mayo scissors. Place a sternal retractor, and widen it to provide adequate visualization of the heart and pericardial sac.
    2. Lift the pericardium with DeBakey forceps, and open the pericardium with Metzenbaum scissors.
    3. Placing the three piezoelectric sonomicrometry crystals:
       1. Make three small 1 mm cuts on the epicardium of the LV, forming the corners of a triangle. Place the piezoelectric sonomicrometry crystals inside the epicardium cuts.
       2. Secure the wires to the cardiac surface with a 5-0 polypropylene U-stitch. When recording using sonomicrometry, pause the mechanical ventilation to allow for an accurate recording over two to three heartbeats.
          NOTE: If the heart fibrillates, 1% lidocaine is not effective, and epicardial defibrillation is needed, turn off the sonomicrometer, and disconnect it from the data acquisition system to protect both from electrical input.
    4. LAD artery isolation:
       1. Encircle the second or third diagonal branch of the LAD artery with a polypropylene suture (3-0) on a taper needle.
       2. Remove the needle, and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.
       3. Place a pledget between the snare and the coronary artery to avoid damaging the coronary artery and/or causing vasospasm with ligation.
       4. Using DeBakey forceps, pick up a rectangular PTFE felt pledget (approximately 7 mm x 3 mm). Place the pledget between the two polypropylene filaments so that it is sandwiched between the isolated LAD artery and the vinyl tube when the snare is tightened.
    5. Measurement of the LVDP:
       1. Place a 5-0 polypropylene U-stitch in the apex of the LV. Make a small 1 mm incision with an 11 blade into the LV apex.
       2. Insert a 3 French balloon catheter into the LV lumen. Secure the catheter to the LV by tying it to the 5-0 polypropylene U-stitch suture.
       3. Connect the catheter to the transducer connected to the monitor to record the LVDP. Record the LVDP using the data acquisition system (described below). Zero the catheter to record the hemodynamic variables by opening the three-way stopcock to the air and zeroing on the monitor.
    6. Data acquisition system
       1. Initiate the data acquisition system (see the Table of Materials) on the computer/laptop being used. Connect the wire from the monitor to the computer/laptop.
       2. Select Channel 1 on the data acquisition system, and name it LVDP. Zero the transducer using the monitor.
          NOTE: If connecting the BP and HR to the data acquisition system, follow the same process: connect the wire to the laptop, select Channel, and zero if measuring the BP.
15. Occlude the coronary artery by tightening the snare by pressing down on the vinyl tube while pulling up on the polypropylene suture filaments. Maintain the desired tightness with a mosquito clamp by directly clamping the tube and fixing it in place.
16. Confirm myocardial ischemia visually by the regional cyanosis of the epicardium. Regional ischemia can also be confirmed on ECG with the presence of an ST segment and T wave changes.
17. After visual confirmation, induce regional ischemia for 30 min under anesthesia.
    1. At 0 min, 10 min, 20 min, and 30 min during the regional ischemia, assess the FS by 2D ECHO for both the survival and non-survival cases.
    2. Assess the LVDP and sonomicrometry continuously during the pre-ischemia time, myocardial ischemia time, and post-ischemic time for the non-survival cases.
    3. If needed, delineate the area at risk by ligating the artery again with the polypropylene suture stitch left in place. Cross-clamp the aorta, and inject Monastral Blue pigment 98% (diluted 1:5 in PBS) through the aorta using a cardioplegia needle. The perfused areas of the myocardium will stain blue, and the area at risk will remain unstained.
    4. Continuously monitor and record the HR, BP, and O₂ saturation.
    5. Allow the animal to recover for 2 h (non-survival) or 28 days (survival).
       NOTE: ECG can be used to confirm reperfusion. Although not seen in the experiment conducted in this study, hypokalemia can often occur during reperfusion and can be corrected with potassium control or an appropriate infusion.
18. Conclusion of the procedure
    1. Survival cases
       1. In survival cases, trim the 3-0 polypropylene thread used for the snare, loosely tie the ends together, and leave it in place. Identify the area at risk and the infarct zone by the 3-0 polypropylene thread.
       2. After the procedure is completed, close the incision in three layers.
          1. Close the first layer by tying two 2-0 polyglactin 910 figure-of-eight stitches around the ribs.
          2. Close the muscular and subcutaneous layers with a 3-0 polydioxanone suture in a running fashion.
          3. Close the skin in a subcuticular fashion using a 5-0 monofilament suture. Use a buried running suture to minimize the irritation felt by the animal.
       3. Evacuate the pleural air by performing a needle thoracentesis.
       4. Apply a fentanyl transdermal patch for 72 h to facilitate the postoperative pain management.
       5. Perform transthoracic echocardiography at the 1 week and 2 week time points postoperatively to assess the trends in the FS.
       6. Following the predetermined recovery period, sedate, intubate, and anesthetize the animal as above. Perform a median sternotomy. Expose and open the pericardial sac. Euthanize the rabbit under deep anesthesia by removing the heart en bloc, allowing the animal to expire by exsanguination.
    2. Non-survival cases
       1. After the experiment and having assured deep anesthesia, completely expose the heart, and remove it en bloc for biochemical and tissue analysis. The animal expires by exsanguination.

Results

Following the protocol (Figure 1), myocardial ischemia was confirmed immediately by the direct visualization of cyanosis of the epicardium.

Standard ECGs (three limb leads: I, II, and III, and three computed augmented leads: aVL, aVR, and aVF) were recorded continuously pre-ischemia, during ischemia, and at reperfusion (Figure 2). The ECGs demonstrate tachycardia, arrhythmias (i.e., ventricular fibrillation), conduction system defects...

Discussion

Our protocol demonstrates a reliable methodology for performing acute regional myocardial ischemia in the rabbit. The left mini-thoracotomy approach is ideal for survival cases, for which the incision and associated pain must be minimized. Importantly, diuretic therapy was not necessary prior to extubation, and there was no mortality intraoperatively in the non-survival group or at 4 weeks postoperatively in the survival group. When the design of the protocol requires a non-survival case, or when more detailed monitoring...

Materials

Name	Company	Catalog Number	Comments
#10 blade	Bard Parker	371210
#11 blade	Fisher Scientific	B3L
22 G PIV needle	BD Insyte	381423
Acepromazine	VETONE	NDC 13985-587-50	0.5 mg/kg IM and IV
Aline pressure bag	Infu-Stat	2139
Angiocath	Becton Dickinson	382512
Arterial Catheter	Teleflex	MC-004912
Atropine	Hikma Pharmaceuticals	NDC 0641-6006-01	0.01 mg/kg IM
Betadine and 70% isopropyl alcohol	McKesson	NDC 68599-2302-6
Blood gas machine	Siemens	MRK0025
Bovie	Valleylab	E6008
Bulldog clamps	World Precision Instruments	14119
Bupivacaine	Auromedics	NDC 55150-249-50	3 mg/kg IM
Butorphanol	Roxane	NDC 2054-3090-36	0.5 mg/kg IM
Clear acetate sheet	Oxford Instruments	ID 51-1625-0213
Clipers	Andis	AGC2
DeBakey forceps	Integra	P6280
Echocardiography machine	Philips	IE33 F1
Electrocardiography machine	Meditech	MD908B
Endotracheal tube	Medline	#922774
Fentanyl	West-Ward	NDC 0641-6030-01	1–4 µg/kg transdermal patch
Formaldehyde solution 10%	Epredia	94001
Glass plates	United Scientific	B01MUHX6MR
Heparin Sodium	Sagent	NDC 69-0058-02	1000U in 1 mL 3 mg/kg
Hot water blanket	3M	55577
Isoflurane	Penn Veterinary Supply, INC	NDC 50989-606-15	1%–3%
Ketamine	Dechra	NDC 42023-138-10	10 mg/kg IV
Lab Chart 7 Acquisition Software	Adinstruments
Lactated Ringer's solution	ICUmedical	NDC 0990-7953-09	10 mL/kg/h
Laryngoscope	Welch Allyn	68044
Left ventricule lumen catheter 3Fr	McKesson	385764-EA
Lidocaine (1%)	Pfizer	4276-01	1–1.5 mL/kg IV
LVDP transducer	Edward	PDP-ED
Marking pen	Viscot	1451SR-100 Unsterile
Mayo scissors	Mayo	S7-1098
Medetomidine	Entireoly Pets Pharmacy	NDC 015914-005-01	0.25 mg/kg IM
Metzenbaum scissors	Cole-Parmer	UX-10821-05
Monastra. Blue pigment 98%	Chemsavers	MBTR1100G
Monocryl 5-0	Ethicon	Y463G
Mosquito clamp	Shioda	802N
PDS 3-0	Ethicon	42312201
Piezoelectric sonomicrometry crystals	Sonometrics	Small 2mm round
Plegets	DeRoyal	32-363
Povuine Iodine Prep Solutions	Medline	MDS093940
Precision vaporized system face mask	Yuwell	B07PNH69BF
Prolene 3-0	Ethicon	8665G
Proline 5-0	Ethicon	8661G
Pulse oximetry probe	Masimo	9216-U
Rib spreader	Medline	MDS5621025
S12 Pediatric Sector Probe	Phillips	21380A
Sonomicrometer	Sonometrics	BZ10123724
Sterile gauze	Medline	3.00802E+13
Sterile towels	McKesson	MON 277860EA
Sternal retractor	Medline	MDS5610321
Sutures for closure	J&J Dental	8698G
Telemetriy monitor	Meditech	MD908B
Temperature probe	Omega	KHSS-116G-RSC-12
Triphenyl tetrazolium chloride (1%)	Millipore	MFCD00011963
Ventilator	MedGroup	MSLGA 11
Vicryl 2-0	Ethicon	V635H
Vinyl tubing	ABE	DISW 3001