Development of a Novel Calcified Total Occlusion Model in<br />
Porcine Coronary Arteries

Chronic total occlusion (CTO) represents 10–20% of all angioplasty cases and remains a challenge for interventional cardiologists.^1–3 CTOs are associated with significant angina, impaired left ventricular function and poor long-term outcomes.⁴ Although new devices have been developed and implemented, the success rate for intervention of CTO is still only up to 55–80%.^4,5 Significant limitations to successful recanalization include the failure to cross the wire through the CTO, the age of occlusion, lesion length, lack of presence of a tapered stump, tortuosity and severe calcification.⁶ Previous reports have demonstrated that successful revascularization of CTOs significantly improves angina in symptomatic patients, improves left ventricular function, and reduces mortality.^7–9 Therefore, it is very important to create a useful large animal model of a CTO, which would facilitate the development of novel devices and techniques to enhance success rate. The purpose of this study was to create a calcified total occlusion model in porcine coronary arteries using a catheter-based technique. This can be utilized in preclinical evaluation of CTO technologies.

Materials and Methods
Experimental study (Day 0). This study was carried out according to the Guide for Care and Use of Laboratory Animals and was approved by the Institutional Animal Care and Use Committee at the Skirball Center for Cardiovascular Research of the Cardiovascular Research Foundation.
A total of twenty Yorkshire swine (24–41 kg) received a beta-blocker (atenolol 25 mg) orally beginning 2 days prior to and continuing for 3 days after the procedure. Each swine was anesthetized with telazol 3–5 mg/kg and xylazine 1–2 mg/kg. An intravenous (IV) line was placed using aseptic technique in the auricular marginal vein, and IV fluids (lactated Ringer’s or 0.9% saline) were administered throughout the procedure. Antiarrhythmics were added to these IV fluids (lidocaine 200 mg/l and metoprolol 5 mg/l). The animals were intubated and ventilated with 98% oxygen and 2% isoflurane. While using general sterile technique, a cutdown procedure was performed to identify and isolate the left common carotid artery. A 9 Fr vascular access sheath was then introduced and secured with suture. Before catheterization, heparin (5,000–10,000 U) was injected to maintain an activated clotting time (ACT) of 250–300 seconds.
Occlusive material. Bone chips were obtained, in advance, from previously euthanized animals. A harvested rib was dried and crushed into a mixture of powder and bone chips. The bone chips were then sterilized and the process repeated as necessary to supply enough material for all animals in the study. Absorbable gelatin sponge (Gelfoam, Pharmacia & Upjohn Company, Kalamazoo, Michigan) was cut into 2 x 20 mm sections and mixed with the sterilized bone at the time of the procedure.

Experimental procedure. Via the cannulated left common carotid artery, the left or right coronary artery (RCA) was cannulated with a 9 Fr JL 3.5 guiding catheter under fluoroscopic guidance. The bone chips with absorbable sponge were preloaded into a 7 Fr hockey stick (HS) catheter and introduced into the 9 Fr guiding catheter. The 7 Fr HS catheter was advanced to the intended site over a 0.014 inch guidewire using fluoroscopic guidance. Using a 5 Fr multipurpose catheter loaded with a 0.035 inch guidewire, the bone chips with absorbable sponge were pushed out from the end of the 7 Fr HS guiding catheter into the coronary artery (Figure 1). After the material was successfully deployed, coronary angiography was performed.
If there was vascular spasm, an intracoronary injection of 100 μg nitroglycerin was administered. Coronary angiography was repeated to confirm the total occlusion. Continuous electrocardiographic and hemodynamic monitoring were assessed continuously during the evolving infarction. Occasionally, ventricular arrhythmias occurred during the first 30 minutes of occlusion. Continuous 0.1 mg/kg/hour metoprolol tartrate (5 mg/5ml) was administered during the procedure to stabilize the arrhythmias and if episodes of nonsustained ventricular tachycardia occurred, an additional 1 mg/ml metoprolol bolus injection was administered.
If the vital signs were stable 60 minutes later, the sheath was removed, the vessel ligated with 2–0 silk suture, and the incision site closed in 2–3 layers with appropriate suture material (typically 2–0 ethilon followed by 3–0 vicryl). After confirmation that there was no oozing blood, the isoflurane was discontinued and the animal was extubated when the gag reflex had returned. Buprenorphine 0.01–0.02 mg/kg intramuscular and flunixin 1–2 mg/kg IV were injected for routine pain management. The animals were also given cefazolin to prevent infections (1 g IV).
Follow-up angiography, sacrifice and histology (day 28). After 1 month, the same preoperative procedure and anesthesia was performed as in the initial procedure. A 7 Fr sheath was placed in the femoral artery by direct puncture and the Seldinger technique. Heparin (5000–10,000 U) was given as necessary to maintain an ACT of 250–300 seconds throughout the procedure. The left coronary artery and RCA were cannulated with a 7 Fr HS catheter under fluoroscopic guidance and angiograms were taken in multiple projections, with particular care to observe for distal contralaterals or bridging collateralization.
Euthanasia. After completion of the study, the animal was euthanized while under general anesthesia by IV injection of pentobarbital euthanasia solution (100 mg/kg) and/or potassium (40 mEq). The heart was excised and the coronary arteries were flushed with lactated Ringer’s solution and then pressure-perfusion fixed with 10% neutral buffered formalin for 30 minutes.
Histology. The occluded vessel was explanted and observed at the gross anatomical level, and then cut into 2–3 mm sections. The specimens were embedded in paraffin and cut on a rotary microtome into 5 μm sections, which were mounted on charged slides and stained with hematoxylin-eosin and elastic Van Gieson.
Intravascular ultrasound imaging. After the 28-day follow up, 1 animal underwent an intervention for the calcified total occlusion. After successful recanalization, intravascular ultrasound imaging (IVUS) was performed using a commercially available system (Boston Scientific Corp., Natick, Massachusetts). The imaging catheter was advanced distal to the occluded segment under fluoroscopic guidance. Using automated pullback (0.5 mm/second), ultrasound images were obtained and recorded digitally on CD-ROM.

Results

Angiographic findings. A total of 20 animals underwent the total occlusion creation with the bone chips and absorbable sponge: 14 in the left anterior descending artery (LAD), 4 in the RCA, and 2 in the left circumflex (LCX) artery. Twelve out of 20 animals survived and underwent follow-up angiography at day 28. In the LAD group, 8 animals survived until sacrifice, while 6 animals experienced sudden death within a day. Within these surviving 8 animals, all had completely occluded vessels, with 4 of them showing right-to-left distal collateralization and 4 of them showing antegrade bridging collaterals. Figure 2 shows one of the calcified total occlusions created in the LAD. The LAD has a total occlusion mid-vessel just after the second diagonal branch, and there was a contralateral collateral from the RCA. Visible calcification under fluoroscopy was apparent on the anterior wall in the left ventriculography images with and without contrast (Figure 3). In the LCX group, 2 animals survived and revealed totally occluded vessels, with small bridging collaterals in both. Figure 4 shows one of the LCX cases with the baseline and 1-month follow-up angiograms. In the RCA group, 2 animals survived, with the other 2 having experienced sudden death during the postprocedure period. In the 2 surviving animals, the vessels recanalized. Figure 5 shows one of the RCA cases. The RCA had a diffuse narrowing in the entire vessel. Table 1 shows detailed information on the 12 surviving animals.

Histological findings. The occluded plaque segments were infiltrated with inflammatory cells and microvessel channels. There was moderate disruption of the internal elastic lamina (IEL), external elastic lamina and medial wall (Figure 6). Hematoxylin-eosin and elastic Van Gieson stainings showed calcified nodules and microchannels within the occlusions, media and adventitia.
Intravascular ultrasound findings. In 1 animal an attempt was made to cross the calcified total occlusion in the LAD. After successful revascularization, IVUS was performed. Figure 7 shows the short- and longitudinal axis images. Superficial calcium is seen at multiple sites between 12 and 3 o’clock on the luminal surface, with acoustic shadows, which indicate that the superficial calcium was within the occlusion.

Discussion

Our study demonstrated reliable and repeatable calcified total occlusions in the porcine coronary arteries. Coronary angiography showed total occlusions, visible calcium under fluoroscopy, and rich collateralization from the bridge or contralateral collaterals. The histology showed total occlusions with calcified nodules and abundant microchannels.
Over the last 30 years, researchers have tried to create animal CTO models in coronary and peripheral arteries. The initial studies were performed with arterial ligation and ameroid constrictors^10,11 to occlude the vessel by surgical techniques. Surgical ligation and ameroid constrictors are external occlusions that are not suitable for the evaluation of CTO devices in the coronary artery. Subsequent endoluminal approaches using conventional interventional techniques were developed to mimic human CTOs. Thrombin injection,^12,13 balloon injury,¹⁴ copper stenting and thermal injury^15,16 were all attempted. The histology showed mature fibrous tissue, inflammatory reaction, neointimal proliferation, multiple small intraluminal vascular channels and disruption of the IEL, which have several characteristics of human CTOs. However, none of them demonstrated severe calcification histologically.

Recently, percutaneous occlusion devices have been developed. Prosser et al reported that deployment of a microporous poly L-lactic acid polymer occluded the coronary artery and was histologically similar to human CTO plaque, including the presence of microvascular channels, dense collagen and elastic tissue within the occlusion.¹⁷ Kipshidze et al also showed successful occlusion using the Biomerix Vascular Occlusion Device.18 Although the delivery methods are similar to our delivery technique, the histological findings did not demonstrate calcified nodules within the occlusions.
The histological characteristics of human CTOs are fibrous tissues, calcification, inflammation and neovascularization.^19,20 Calcifications are more prevalent in hard plaques and the extent and severity of the calcification relate to the age of the CTO.²¹
Our study showed that the occluded vessel segment exhibited calcified nodules, neointimal proliferation, inflammatory cells, abundant microchannels and rich collateralization from the bridge or contralateral collaterals, all of which are similar to human CTOs.⁶
Furthermore, we performed intervention for the calcified total occlusion in one of the animals. After successful recanalization, IVUS was performed from the distal LAD through the occluded segment. The IVUS image demonstrated superficial calcification in the occluded segment. The images of the calcium are similar to what is seen in human IVUS images.22 In terms of the hardness of the lesion, the feedback from the end of the stiff wire was similar to human calcified CTOs. Based on histology, angiography and IVUS, we were able to successfully mimic human CTOs for use in CTO device evaluations.
Study limitations. These studies are clearly preliminary and a number of limitations remain. Only 1 month passed between creation and evaluation of the occlusion, as opposed to the slower development of true CTOs found in humans. Further studies are needed to evaluate subjects such as the development of a tough fibrous cap, dense fibrous tissue, calcification and collateralization over the long term. Moreover, there was a high mortality rate from acute infarction after material delivery. There were no successful CTO creations in the RCA. We suspect the volume of the occlusive material was not sufficient to sustain the occlusion due to the large diameter of the RCA in swine. Additional studies are needed to improve the mortality rate as well as the success rate in the RCA, and to evaluate histological changes over a longer survival period.

Conclusions
To our knowledge, this is the first severe calcified total occlusion in a porcine coronary model. We were able to successfully create reliable and repeatable calcified total occlusions in the porcine coronary arteries (LAD and LCX). Coronary angiography showed total occlusions, visible calcium under fluoroscopy, and rich collateralization from the bridge or contralateral collaterals. The histology showed total occlusions with calcium and microchannels, which is similar to CTOs in human coronary arteries.
This method can be utilized in many preclinical evaluations of CTO technologies such as new coronary wires, debulking devices, crossing devices and even physician training.

References

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