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Ann Thorac Surg 2006;82:1559-1566
© 2006 The Society of Thoracic Surgeons

Reviews

Controversies in the Use of Intraluminal Shunts During Off-Pump Coronary Artery Bypass Grafting Surgery

Sathiakar Paul Collison, DNB*, Anil Agarwal, MCh, Naresh Trehan, MD

Escorts Heart Institute and Research Centre, New Delhi, India

* Address correspondence to Dr Collison, Escorts Heart Institute and Research Centre, Okhla Rd, New Delhi, India 110025 (Email: spcollison{at}gmail.com).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Conclusions
 References
 
Technical advances have made the performance of multivessel off-pump coronary artery bypass feasible. Snaring and intraluminal shunts are the techniques used for vascular control. Snaring provides a bloodless surgical field, is usually well tolerated by the patient, and is supported by years of clinical experience. Intraluminal shunts aim to achieve hemostasis at the arteriotomy site and to allow antegrade flow to provide myocardial protection. There are unresolved issues regarding whether shunts have a clinical benefit, do provide adequate flow to provide myocardial protection, and whether they cause significant endothelial damage. In this article, we have reviewed the literature to lend perspective to these issues.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Conclusions
 References
 
Technical advances have made multivessel coronary artery bypass without the use of cardiopulmonary bypass feasible. The challenges to the surgeon performing off-pump coronary artery bypass graft surgery (OPCABG) lie in the choice of incision, exposure of target vessels, control of motion of the heart, vascular control, and myocardial protection. Vascular control has been performed either using extraluminal occlusion by vessel loops or sutures (hereafter termed snaring) or by using an intraluminal shunt (ILS). Snaring techniques were the first to be introduced into the field of OPCABG. Although described in 1975 [1], it is only recently that the ILS has become widely available.

Surgeons who use snaring claim that the technique is easy to apply; that it provides a completely bloodless field enabling precise anastomosis; that the short period of obligatory ischemia is usually well tolerated; that endothelial damage caused by an ILS can be avoided; that shunt insertion is difficult; and finally, that several years of clinical experience with snaring supports its use.

Proponents of ILS usage claim the advantages are as follows: it provides a relatively bloodless field; the forward flow in the shunt helps prevent ischemia, facilitating stabilizer placement and positioning of the heart; it facilitates the construction of the anastomosis by protecting the back wall of the artery; traction on the ILS exposes the edges of the arteriotomy well; and it facilitates training of surgeons in OPCABG [2].

In an attempt to answer the question "Should we snare or shunt during OPCABG?" we have reviewed the literature to answer the following questions: Is there clinical benefit to the use of an ILS? How much endothelial damage does an ILS cause? What is the appropriate size of an ILS that should be used? How much blood flows through a shunt? How does active coronary perfusion compare with ILS? And finally, what is the effect of an ILS on graft patency?


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Conclusions
 References
 
The scientific literature of the English language was reviewed by searching Medline from 1966 through March 2006. The keywords used in the search included "shunts," "intraluminal shunts," "snare," "off-pump coronary artery bypass," "endothelial injury/damage," "myocardial protection," "coronary active perfusion," and "perfusion-assisted direct coronary artery bypass." The search terms were combined using the Boolean operator term "or" to find all abstracts pertaining to the chosen search terms. These individual terms were then combined using the Boolean operator term "and" to find articles that contained information of all search terms [3]. The reference lists of articles found through these searches were also reviewed for relevant articles. Links provided on the web sites of published articles were searched for relevant articles. The primary search yielded 168 articles. Of these 96 were excluded as they pertained to noncoronary surgery, angioplasty, or diagnostic techniques. Further, case reports and studies of single-surgeon experiences were excluded.

Is There Clinical Benefit to the Use of ILS?
Dapunt and colleagues [4], Gandra and Rivetti [5], and Sepic and associates [6] have reported experimental evidence of the benefit of ILS in pigs (Table 1). In each of these studies, there were two groups of pigs undergoing anastomosis of left internal mammary artery to left anterior descending artery—in one group snare was used, and in the other ILS was used. Dapunt and colleagues [4] found that with snaring, there was decreased cardiac output during anastomosis and extending into the reperfusion period, along with depressed global and regional left ventricular (LV) function, suggesting myocardial stunning. They performed myocardial biopsies that revealed that when ILS was used, there was less liberation of myocardial heat shock protein 70 (an indicator of ischemia) and better preservation of myocardial adenosine triphosphate. Gandra and colleagues [5] studied epicardial monophasic action potentials and biochemical tests as markers for ischemia and found more evidence of ischemia in the animals that were snared. Sepic and colleagues [6] studied regional myocardial function using piezo-electric ultrasonic crystals placed subendocardially and a sonomicrometer to assess the functional change in area; they also studied regional myocardial blood flow (RMBF) using radiolabelled microspheres. They were able to show that with ILS, RMBF and functional change in area were maintained in the normal range.


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  		Table 1. Studies Comparing the Clinical Benefit of Snaring or Intraluminal Shunt (ILS)
 
Two clinical studies have compared snaring with ILS in patients undergoing isolated left internal mammary artery to left anterior descending artery anastomosis. Luchetti and colleagues [7] found preserved LV wall motion score index and preserved ejection fraction determined by transesophageal echocardiography when ILS was used. Using a minimally invasive approach, Menon and colleagues [8], compared the techniques using hemodynamic data and transesophageal echocardiographic measurements and found reduced ischemia and preserved LV function when ILS was used.

The only study [9] that has compared snaring with ILS during multivessel OPCABG involved the randomization of 40 patients into two groups who underwent OPCABG using either snare or ILS. All had critical triple-vessel disease and ejection fraction greater than 40%. Patients with recent myocardial infarction, emergency operation, and reoperations were excluded. The investigators compared hemodynamic data, intraoperative events, and postoperative outcome between the two groups. They could show a small but statistically significant improvement in hemodynamic variables during the anastomosis in the group in which ILS was used. In the snared group, there was a greater delay in restoration of hemodynamic parameters to baseline. However, there was no significant difference in mortality, anastomotic time, perioperative myocardial infarction, need for inotropes, or length of intensive care unit stay between the two groups.

Thus, there are data to suggest that during the anastomosis, the use of ILS does provide a measure of benefit by way of better hemodynamics, better LV function, and less myocardial ischemia. There may be a protective effect against myocardial stunning. In contrast, snaring worsened cardiac function, and recovery was delayed. Importantly, there was no difference in the overall clinical outcome between the two techniques. However, most of the data discussed above were derived from low-risk patient populations that would be predicted to do well using either technique. Clearly, randomized clinical studies comparing the techniques are needed in all subgroups of patients, for example, those with low ejection fractions, congestive heart failure, recent infarctions, or redo bypass procedures. These studies may resolve the issues of whether sequential snaring of multiple coronary targets results in a cumulative ischemic insult, or whether the flow allowed by the ILS makes the heart more tolerant of the positioning maneuvers mandated when an OPCABG is performed.

How Much Endothelial Damage Does an ILS Cause?
There are data to suggest that the use of ILS is associated with mechanical endothelial denudation leading to endothelial dysfunction (Table 2). This is a complex issue. A complete discussion of endothelial injury and repair is beyond the scope of this article, but excellent reviews are available [10].


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  		Table 2. Studies of the Endothelial Effects of Snare and Intraluminal Shunt (ILS)
 
Endothelial injury and dysfunction is not unique to the use of ILS. Any contact with the endothelium damages it. It may occur with snaring [11], use of mist-blower [12], papaverine solutions [13], or application of a bulldog clamp on the left internal mammary artery [14]. During on-pump CABG, coronary endothelial reactivity is altered [15], perhaps owing to postischemic impairment of microvasculature [16]. Currently, coronary stents, angioscopy, and coronary brachytherapy devices are being inserted with low-pressure balloons; here too, endothelium is damaged [17]. Hence, it is difficult to avoid endothelial injury. This endothelial loss may be healed by regeneration of the endothelium [18]. Nevertheless, regenerated endothelium is also dysfunctional, having lost some ability to release endothelium-dependent relaxing factors [19]. Hence, it is plausible that vasospasm and atherosclerosis may be accelerated in these areas covered by regenerated endothelial cells. This is the concern of those surgeons who do not use ILS, but it is unproven. It must be emphasized that the studies [20, 21, 22] supporting this argument were experiments of acute endothelial injury in arteries not burdened with preexisting atherosclerosis. Also, in the studies by Hangler and coworkers [20] and Wippermann and colleagues [21], there is a limitation (see section on shunt size) that has affected their results.

To obtain chronically diseased vessels, Perrault and coworkers [23] percutaneously balloon denuded the coronary arteries of pigs, 30 days before submitting them to OPCABG when they compared ILS, snare, and bulldog clamp by tests of vascular reactivity. Surprisingly, they found no difference among the three techniques in endothelium-dependent relaxation. Similarly, the contractile function of the vessel was not affected by any technique, demonstrating the integrity of the underlying smooth muscle cells. Wippermann and colleagues [21] applied either snare or ILS to healthy pig arteries. Three months later, the pigs were sacrificed, and the coronary arteries were examined by electron microscopy. They showed that intimal lesions occur with both techniques. However, animals in the snare group exhibited injuries significantly more often and more severely. They concluded that chronic intimal integrity may be better preserved using ILS. A point to note is that the endothelium in patients with coronary artery disease is already dysfunctional [24]. Perrault and colleagues [23] have argued that in coronary arteries that are already chronically dysfunctional, perhaps additional endothelial damage has limited effect.

It is useful to consider the potential mechanism of injury in the two techniques. In general, snaring involves encircling target vessels with elastic silicone tapes or polypropylene sutures buttressed with silicone tubing and wrapped with tourniquets to obtain hemostasis at the arteriotomy site. These sutures are placed deep into the myocardium with the aim that the subjacent epimyocardial tissue acts as a buffer between the snare and the vessel wall. Still, there is an almost circumferential compression being exerted on all the layers of the vessel wall. Indeed, Gerola and colleagues [25] have shown medial fractures and buckling of elastic lamellae with snaring in a region of diseased coronary artery. Stress and damage to the medial and adventitial layers could potentially induce restrictive arterial remodeling [26]. Adventitial myofibroblasts contribute to neointimal formation [27]. Although earlier work in pigs by Perrault and colleagues [28] indicated the preservation of endothelium with snaring, Hangler and colleagues [11] have shown in humans the damage to the wall by snaring. The necessity of blind application of the snare can cause atheromatous fragments to embolize [29], cause infarction [30], create fistulae between adjacent structures [31], or induce lesions at the site of application [32].

With ILS, damage to endothelium could occur as a result of brushing of the endothelium, leading to its denudation upon ILS insertion. That is perhaps unavoidable. Alternatively, the bulbous ends of the ILS could exert pressure on the wall, leading to endothelial ischemia if too large a shunt is used. Potentially, coronary artery dissection is possible, although this has not yet been reported. There are no effects on the medial and adventitial layers.

From the available data, it is clear that both techniques can damage the vessel wall. Each technique has its own mechanism of injury. The intraluminal shunt may damage endothelial and subendothelial layers, whereas snaring could affect all the layers of the wall. However, the significance of any of the observed effects in dysfunctional coronary arteries is yet to be established.

What Is the Appropriate Size of ILS To Be Inserted?
Selection of an appropriate-sized ILS is of paramount importance. The surgeon would like to have as large an ILS as possible to maximize antegrade flow to obtain hemodynamic benefits, while avoiding creating severe endothelial injury.

The biggest limitation of both the Wippermann [21] and the Hangler [20] studies lies in their selection of shunt size. While Hangler inserted shunts that were "leak proof," in Wippermann's study, the ILS was "slightly oversized" to the coronary artery. Thus, significant endothelial loss was to be expected both from the brushing effect at insertion as well as from pressure on the wall of the artery. That cannot be recommended as the method of using an ILS. Most OPCABG surgeons who use ILS would not attempt to "match" shunt and coronary artery size.

The issue of the appropriate size of an ILS has been studied in the laboratory by Demaria and colleagues [34]. In a study performed in normal pig coronary arteries, they compared coronary morphology and endothelial reactivity after the insertion of ILS of three different sizes with the aim of oversizing, undersizing or using an ILS congruent to the artery diameter. They also assessed the amount of bleeding at the anastomosis site. They found severe decreases in coronary reactivity and a total disappearance of the endothelium with an oversized shunt that provided perfect hemostasis. No significant endothelium loss or dysfunction was noted with a drastically undersized shunt, but continuous bleeding was noted. When a congruent shunt was used (2-mm shunt for 2.2-mm vessel), only selective endothelial dysfunction involving Gi-protein–mediated relaxation was deranged. Further, 50% of the endothelium was preserved. There was intermittent bleeding, but the authors considered that anastomosis could still be done. Wippermann and colleagues [35] used a 1.5-mm ILS in normal pigs and found that only superficial abrasions were produced.

Clearly, the use of an oversized ILS to attain perfect hemostasis during the anastomosis cannot be supported. In the above study [34], whereas the undersized ILS was 62% of the coronary diameter, the congruent shunt was 90% of the diameter. More clinical and experimental studies are warranted to define the optimal ratio of shunt size to coronary diameter. Although this is unproven, in the clinical setting perhaps selecting an ILS that is 75% of the target size (1.5 mm for 2-mm target) would provide the ideal balance between the need for hemostasis and avoidance of endothelial damage, while providing adequate flow through the ILS.

How Much Blood Flows Through a Shunt?
It is intuitive to surgeons who use ILS that when it is in place, there is distal perfusion; and that provides some amount of myocardial protection. Most would have observed good flow of blood out of the distal limb when the proximal limb is inserted. It is also common for ischemic changes that may appear on the electrocardiogram to often resolve when the ILS has been successfully inserted. However, at times, not even the presence of ILS can prevent continued ischemia and hemodynamic instability requiring conversion to cardiopulmonary bypass.

Studies designed to measure the flow through an ILS are difficult to perform. The ILS is inserted distal to a stenotic lesion, the severity of which varies in each case. In the laboratory, too, creation of discrete coronary lesions is difficult. Techniques for measuring regional myocardial metabolism and RMBF in real time, distal to ILS insertion, are not available.

To overcome these difficulties, Kamiya and colleagues [36] investigated the efficacy of the ILS in a theoretical model based on fluid mechanics. Based on the assumption that the intracoronary pressure would be equal both upstream and downstream of the ILS and assuming blood to be a Newtonian fluid, they used equations derived from physics to quantitate flow, in situations of laminar and turbulent flow, before and after ILS insertion. They derived "flow ratio," the ratio of postattachment to preattachment flow. They found that with laminar flow, the flow ratio is proportional to the sixth power of the inner diameter of the ILS; for turbulent flow, flow ratio is proportional to the third power of the inner diameter of the ILS. They found that although commercially available ILS come in sizes of 1 mm to 4 mm, corresponding to coronary artery diameters, in reality their inner diameters are much smaller and, hence, flow ratios are small. Thus, after ILS insertion, coronary flow decreases to 2% to 14% of preattachment flow during turbulent flow and to less than 0.1% in laminar flow. It has been shown that flow is laminar proximal to a coronary stenosis and turbulent distally [37], which is where an ILS is placed. Therefore, it appears that the ILS would carry only 2% to 14% of blood forward. The same group [38] later further corrobated their findings. Using a thermal diffusion technique developed by them to calculated RMBF, they were able to show, in pigs, that after ILS insertion, RMBF decreased to 31% in the epicardium and to 33% in the endocardium. The observed difference in values between the theoretical and animal experiment they attributed to acute recruitment of collaterals. Muraki and coworkers [39] in a different model have shown a fall in RMBF to 30% of baseline under normotensive conditions, and a fall to 10% during hypotensive episodes.

It is evident that the ILS allows only a small fraction of blood forward. It is inserted into a coronary artery with a native stenosis that reduces flow, and the introduction of ILS reduces this flow to approximately 30% of the baseline. Nevertheless, there is usually minimal biochemical or electrocardiographic evidence of ischemia during OPCABG. Indeed, thousands of such procedures have been performed without untoward events using snaring that allows for no forward flow. That is because there are certain protective mechanisms in place that protect the myocardium during the 5 to 10 minutes of warm ischemia needed to perform the anastomosis. These include the formation of collaterals, the possible hibernation of the myocardium, and the distal positioning of the arteriotomy that may limit the amount of myocardium at risk. Still, the temporary occlusion of a coronary artery must be viewed as an ischemic event. Gurbuz and colleagues [40] randomly assigned two groups of patients undergoing OPCABG into snare or ILS groups and found postoperative troponin I levels higher in the snare group.

How much is the barest minimum of blood flow to prevent myocardial ischemia? Unfortunately, there is no definite answer to this question. As discussed nicely by Levinson [41], prevention of ischemia may not require much flow at all. In the early days of angioplasty, failed procedures with ischemia were managed by passing a Stack catheter distally. This autoperfusion catheter was a fine multiperforate tube, and hence, flows were small. But even these small flows were enough in most cases to buy the time needed to perform emergency surgical revascularization. The ILS may be compared with the Stack catheter, and therefore, the small amount of flow allowed may be adequate for the prevention of ischemia.

Previous data [42] indicate that approximately 40 to 60 mL/min of blood is needed for adequate myocardial protection, depending on the mass of myocardium. Grunenfelder and colleagues [43] have studied the pressure-flow relationships of commercially available ILS in an in-vitro model. They have shown that at a pressure of 75 mm Hg, the flow through a 1.5-mm shunt was 40 mL/min. They could also demonstrate correspondingly higher flows with larger shunts. Under hypotensive conditions (pressure 40 mm Hg), flows dropped to 50% of baseline in ILS smaller than 2 mm, and to 70% of baseline for ILS larger than 2 mm. However, even during hypotension, for smaller than 2 mm ILS, flows remained in the range of 10 to 20 mL/min. These data indicate that at a pressure of 75 mm Hg, a 1.5- to 2-mm ILS may very well provide adequate myocardial protection.

Thus, a small amount of myocardial perfusion is allowed by the ILS, and the performed studies emphasize the importance of maintaining adequate blood pressure to allow flow through the ILS. Although only a small fraction of preattachment flow, this amount of blood may be significant in the prevention of myocardial ischemia. Still, further studies are needed to define what minimal level of blood flow provides adequate myocardial protection, and to assess whether the ILS can reliably provide this flow in the operating room.

How Does Active Coronary Perfusion Compare With ILS?
Initial attempts at providing distal perfusion during OPCABG involved using a passive external shunt from the femoral artery or the aorta [44]. However, similar to ILS, it was not known how much perfusion remained when the blood pressure dropped during episodes of hypotension during OPCABG. Therefore, methods of obtaining stable distal perfusion independent of the blood pressure were studied.

Perfusion-assisted direct coronary artery bypass (PADCAB) involves an extracorporeal circuit connected to an in-line servo pump that pumps blood into the distal coronary bed [45]. Initially, inflow to the pump was from the aorta [46], and injection was made into the proximal end of the graft after distal anastomosis. More recent modifications use femoral cannulation and small caliber cannulas designed to allow perfusion during anastomosis [47]. The circuit is designed to deliver flow at supraphysiologic pressures to allow perfusion independent of coronary resistance, and to allow intracoronary injection of additives like nitroglycerine and adenosine [47, 48]. Muraki and colleagues [39] have provided experimental evidence of the value of a similar system in pigs. They compared ILS with an intra–left anterior descending artery cannula connected to a pump that enabled distal perfusion. They studied RMBF, oxygen consumption, lactate extraction, and systolic shortening with either technique. They found significantly impeded blood flow with the ILS in place, which worsened to the threshold of ischemia during hypotensive episodes and was associated with reduced oxygen consumption and myocardial function. In contrast, oxygen variables and RMBF exceeded baseline values with active perfusion.

Cooper and colleagues [49] have presented the clinical results of 169 PADCAB procedures compared with 358 patients undergoing routine OPCABG. In general, the PADCAB patients were sicker and had worse coronary artery disease. The PADCAB group received 3 mg/kg heparin, and nitroglycerin was added to the circuit to maintain perfusion pressure of 100 mm Hg. They found that there was a significantly greater number of grafts and a higher number of lateral wall grafts in the PADCAB group than the OPCABG group. The system provided 88 mL/min of coronary flow. There was a trend to more blood transfusions and longer hospital stay in the PADCAB group.

Contemporaneously, in Japan, Kamiya and colleagues [50] reported their system that ensures a physiologic blood flow synchronized with the heartbeat during anastomosis using a syringe pump system. To validate their coronary active perfusion system (CAPS), they compared native coronary flow, passive femoral artery shunt, and CAPS in pigs. They showed that with CAPS, coronary flow was 13 mL/min versus 5 mL/min for femoral shunt at 0.1 mL/stroke and 42 mL/min versus 33 mL/min at 0.4 mL/stroke at a coronary perfusion pressure of approximately 65 mm Hg. They then compared CAPS and snaring with regard to ventricular arrhythmias, myocardial oxygenation, and RMBF in pigs [51], using a CAPS flow rate of 0.1 mL/stroke that provides approximately 15 mL/min of flow. They found preservation of RMBF, oxygenation, and hemodynamic variables, and protection against arrhythmias when CAPS was used. They emphasized that myocardial nutrition could be maintained at these low flow rates.

To refine their CAPS system, they studied the effect of supplying CAPS at 4 different time periods in the cardiac cycle (one systolic, three diastolic) [52]. RMBF, LV function and normal myocardial histology were better maintained with diastolic versus systolic injection. They have postulated that if flow is pulsatile and diastolic in nature, then perhaps smaller flow rates are adequate even at coronary perfusion pressure of 50 to 65 mm Hg. They have reported their experience in 524 consecutive patients undergoing OPCABG using this system [53]. Salient features of their surgical technique include heparinization therapy to maintain activated clotting time above 200 seconds, titration of CAPS according to myocardial oxygenation measured by near-infrared spectroscopy, and a 3-minute test snaring with the perfusion cannula in place. They performed 1,583 anastomoses (3.03 per patient) with an event rate during anastomosis of 0.6% (3 of 524 with multifocal ventricular ectopy). Two patients had low output, and 2 had postoperative infarctions. Angiograms performed at 2 weeks in 73% of patients showed patency rates in the region of 99% for all grafts.

Only one clinical trial has compared perfusion methods, and it was presented by Vassiliades and colleagues [54]. They randomly assigned 151 consecutive unselected patients to either receive no perfusion, passive perfusion (aortocoronary shunt), or PADCAB. There were no exclusions based on LV function, anatomy, or comorbid risk factors. There was no difference in intraoperative variables, grafts performed, inotrope usage, blood transfusions, or postoperative outcomes among the groups. Analysis of high-risk subgroups also failed to reveal any differences. However, there was a statistically higher level of troponin I at 24 hours in the passive perfusion group as compared with PADCAB.

Thus, there are no data comparing ILS with active perfusion systems. The PADCAB system involves an extracorporeal circuit, nonpulsatile flow, full heparinization, and supraphysiologic perfusion pressures. In contrast, the CAPS system is pulsatile, synchronized with diastole, and provides approximately 15 mL/min of blood at perfusion pressure of 50 to 65 mm Hg. Further studies are needed to define the role of these systems in OPCABG.

What Is the Effect of ILS on Graft Patency?
Some surgeons [2, 6] believe that ILS facilitates precision in performing the anastomosis. Traction on the string of the ILS excellently presents the margins of the arteriotomy while the body of the shunt prevents inadvertent suturing of the back wall. An appropriately sized shunt provides space between the ILS and vessel margin that facilitates suturing. The presence of the ILS may also reduce the adverse endothelial effects of the gas-blower. Smooth removal of the ILS is also proof of anastomotic patency. That could have implications on graft patency.

However, there are no data available regarding the effect of ILS usage on graft patency. Parolari and colleagues [55] recently conducted a meta-analysis of randomized trials comparing OPCABG with CABG graft patency. They could find five comparable studies. Unfortunately, in all of these studies, snaring was the technique used. Graft patency was inferior in those undergoing OPCABG. Would the use of ILS have improved the graft patency? That is an intriguing question, and only a randomized trial comparing snare with ILS would provide an answer.


   Conclusions
Top
 Abstract
 Introduction
 Material and Methods
 Conclusions
References
 
So should we be using a snare or ILS? Many issues remain unresolved, and techniques are in evolution [56–59]. Snaring has withstood the test of time. Still, there are clinical benefits to the use of ILS during anastomosis, and there is evidence that there is significant flow through the shunt. A properly sized shunt can minimize endothelial damage even though the significance of these lesions in dysfunctional vessels is unknown. Damage to the media and adventitia is avoided. Ultimately, we believe the knowledge of superficial endothelial injury should not prevail over the wisdom of allowing antegrade flow in every patient undergoing OPCABG.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Conclusions
 References
 

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