Contemporary assessment of foot perfusion in patients with critical limb ischemia

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

Available online at www.sciencedirect.com

www.elsevier.com/locate/semvascsurg

Contemporary assessment of foot perfusion in patients with critical limb ischemia Erik Benitez, Brandon J. Sumpio, Jason Chin, and Bauer E. Sumpion Department of Vascular Surgery, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510

article info

abstra ct Significant progress in limb salvage for patients with peripheral arterial disease and critical limb ischemia has occurred in the past 2 decades. Improved patient outcomes have resulted from increased knowledge and understanding of the disease processes, as well as efforts to improve revascularization techniques and enhance patient care after open and endovascular procedures. An imaging modality that is noninvasive, fast, and safe would be a useful tool for clinicians in assessing lower-extremity perfusion when planning interventions. Among the current and emerging regional perfusion imaging modalities are transcutaneous oxygen monitoring, hyperspectral imaging, indocyanine green dye
based fluorescent angiography, nuclear diagnostic imaging, and laser Doppler. These tests endeavor to delineate regional foot perfusion to guide directed revascularization therapy in patients with critical limb ischemia and foot ulceration. & 2014 Elsevier Inc. All rights reserved.

1.

Introduction

The most important factor for determining the healing potential of a pedal wound is the degree of perfusion to the affected segment of the foot. Traditionally, the gold standard in the treatment of critical limb ischemia (CLI) has been open bypass surgery for any patient able to tolerate the procedure for restoration of perfusion. For patients who are not candidates for open revascularization due to medical comorbidities or lack of an appropriate target or outflow vessels, endovascular interventions have provided the ability to restore arterial flow in the affected extremity and, in many instances, has become the first-line option. The decision making for intervention involves history taking, physical examination, and review of both physiologic

n

Corresponding author: E-mail address: [email protected] (B.E. Sumpio).

http://dx.doi.org/10.1053/j.semvascsurg.2014.12.001 0895-7967/$ - see front matter & 2014 Elsevier Inc. All rights reserved.

markers and anatomic imaging obtained through noninvasive imaging (Table 1). Vascular surgeons have traditionally approached this task of revascularization, whether through open bypass surgery or endovascular intervention, through the concept of global perfusion with the “best vessel” approach for either bypass or endovascular interventions. The target outflow artery is chosen based on technical suitability, disease characteristics, length of bypass required, conduit available, and patent of runoff vessels. However, there have been several contemporary studies evaluating the rate of secondary amputation in patients with CLI, despite interventions via open bypass or endovascular interventions, which demonstrate that up to 18% of cases have persistent ischemic ulceration despite “successful” revascularization [1,2].

4

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

Table 1 – Overview of global perfusion studies. Modality

Description

Doppler (physiological)

Continuous wave Doppler transmits and receives sound waves to evaluate rate of blood flow in vessels

ABI/segmental pressure (physiological)

Measuring the difference in blood pressure between the brachial and ankle arteries with Segment pressures displaying a gradient if there is PAD Evaluates and records variations in the volume or blood flow through an extremity as well as arterial pulsatility

Plethysmography/ PVR (physiological)

Ultrasound (anatomical)

Sonography to visualize vessel caliber, obstruction, flow, and characterize plaque lesions

CTA (anatomical)

CT
cross-sectional imaging to provide 360 reconstruction of vasculature

MRA (anatomical)

MR
cross-sectional imaging to provide 360 reconstruction of vasculature

Benefits

Limitations

Fast, noninvasive, cost effective Office/clinic application Fast, noninvasive, cost effective Office/clinic application Fast, noninvasive, cost effective Office/clinic application Fast, noninvasive, cost effective Office/clinic application Fast and noninvasive More cost effective vs traditional angiography Noninvasive Not obscured by vessel calcification

Limited by user skill and patient body habitus Cannot localize location of obstruction Can be false elevated secondary to arterial calcinosis in DM and renal disease Must be combined with PVR and Segmental pressures to provide a relevant and significant clinical information Limited by user skill Difficulty assessing perfusion in distal and smaller size vessels in lower leg and foot Iodinated contrast is nephrotoxic Imaging obscured by vessel calcification Length and cost of study Gadolinium is nephrotoxic Imaging obscured by venous artifact

ABI, ankle-brachial index; CTA, computed tomography angiography; DM, diabetes mellitus; MRA, magnetic resonance angiography; PVR, pulse volume recording; SPECT, single photon emission tomography.

Due to the persistent rate of limb loss despite revascularization via the best vessel approach, there has been increasing interest in performing targeted reperfusion interventions to improve rates of limb salvage and decrease rates of secondary complications. The angiosome concept, introduced by Taylor and Palmer more than 25 years ago, has been a subject of increasing interest [3]. The angiosome was first described by Taylor et al [3,4] and extended to the foot by Attinger and colleagues [5,6], as a three-dimensional perfusion model based on individual segments in the body that follow specific arteriovenous bundles that allow preferential strategies for tissue reconstruction and revascularization. The angiosome consisted of all tissue layers from the skin to the bone. The skin was primarily supplied by direct cutaneous arterioles, with great variance to their density and caliber, which were reinforced by small indirect vessels that were the terminal branches of arteries that supply the deeper tissues. In between each angiosome, there were zones that contained both reduced-caliber arteries (“choke”) and similar caliber arteries (“true”) that anastomosed to form redundant conduits [7]. This allows a given angiosome to receive blood flow from an adjacent neighboring angiosome if the primary source artery is compromised (Fig. 1). There have been various studies comparing outcomes for both open bypass and endovascular interventions using angiosome-based revascularization (direct) versus nonangiosome-based revascularization (indirect) [8]. Soderstrom et al [9] evaluated a total of 250 consecutive legs with diabetic foot ulcers in 226 patients who had undergone infrapopliteal endovascular revascularization. They divided the legs into two groups, depending on whether the inter-

vention was applied directly to an artery supplying the region of the foot where the ulcer was identified, based on the angiosome model, or to feet that were revascularized using the traditional best vessel approach, and compared of ulcer healing time. Directed intervention to the artery supplying the angiosome perfusing the ulcer area was performed in 121 legs and compared with indirect revascularization in 129 legs. Foot ulcers treated with angiosome-targeted infrapopliteal percutaneous transluminal angioplasty (PTA) were found to heal better; the ulcer healing rate was 72% at 12 months for the direct group, compared with 26% at 6 months and 45% at 12 months for the indirect group. Similar results were found by Iida et al [10], who reported a retrospective study in which 369 limbs from 329 consecutive patients with ischemic ulceration or gangrene, or both, and isolated below the knew lesions who underwent endovascular interventions with either angiosome-directed or best vessel indirect revascularization. Post intervention, the patients were followed up at 1 week, 1, 3, and 6 months, and every 3 months thereafter. The overall limb salvage rate was 81% (300 of 369), and the reintervention rate was 31% (114 of 369). The rate of amputation-free survival was 49% in the direct group versus 29% in the indirect group, and the rate of patients avoiding a major amputation post intervention was 82% in the direct group versus 68% in the indirect group. Successful interventions defined as not requiring major or minor amputation were significantly higher in the direct group than in the indirect group for up to 4 years after their index revascularization procedure. As a result of several other studies confirming similar results, there has been increased interest in the development

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

3.

Fig. 1 – Angiosomes of the foot. In the lower extremity, there are six identified arteries based on the three main arteries to the foot: the anterior tibial artery (ATA), three by the posterior tibial artery (PTA), and two by the peroneal artery (PA). The ATA gives rise to the dorsalis pedis artery, supplying the anterior compartment and dorsum of the foot (pink). The PTA gives rise to the calcaneal branch, supplying the medial ankle (black) and plantar heel (green); the medial plantar branch, supplying the medial instep (yellow); and the lateral plantar branch, supplying the lateral and plantar forefoot (blue). The PA supplies the lateral ankle and plantar heel (red and green overlap) via the lateral calcaneal artery, and the anterior ankle via its anterior perforator (pink overlap). Note the overlap of the heel by both the medial calcaneal branch of the PTA and the lateral calcaneal branch of the PA [8]. of effective diagnostic and prognostic studies to evaluate and monitor the regional (angiosome) perfusion of the affected extremity as the current modalities (Table 1) only provide a global assessment of the state of perfusion in the affected extremity. The adaption of the angiosome model as well as utilizing perfusion-based imaging studies would allow the vascular specialists to refine our current understanding of the disease process in CLI while enhancing therapeutic modalities, clinical decision making, and improving outcomes after revascularization interventions.

2.

Assessment of regional foot perfusion

The ideal imaging modality should be applicable to use in both planning a direct-targeted intervention and surveillance of the perfusion in the angiosome after the index procedure. Second, this imaging modality should be able to produce a clearly delineated wound topography as well as targets for angiosome-directed interventions. Lastly, the imaging modality would ideally be dynamic, safe, fast, and easily repeatable in both the intervention and surveillance settings. There are a number of physiologic and imaging modalities that attempt to provide information on regional perfusion of the foot (Table 2). Some of the newer modalities have not been investigated in detail in patients, but have promising results in animal models.

27 (2014) 3–15

5

Transcutaneous oxygen monitoring

Transcutaneous oxygen monitoring, more specifically, transcutaneous partial pressure of oxygen (TcPO2) measurement, provides information regarding local tissue perfusion and skin oxygenation. This technology has been studied to noninvasively assess the healing potential of lower-extremity ulcers or amputation sites as early as 1982, in a seminal article by White et al [11] that demonstrated the utility of TcPO2 in patients with severe peripheral arterial disease (PAD) and CLI before and after undergoing revascularizations, as well as assessing amputation healing potential. The test is performed with platinum oxygen electrodes placed on the chest wall and legs or feet. The absolute value of the oxygen tension at the foot or leg, or a ratio of the foot value to chest wall value, can be used. A normal value at the foot is 60 mm Hg and a normal chest/foot ratio is Z0.9. Local edema, skin temperature, emotional state (sympathetic vasoconstriction), inflammation, and pharmacologic agents limit the accuracy of the test. This was reported by McPhail et al [12], who evaluated the predictive value of wound healing by measuring the TcPO2 in surgical wounds pre- and postoperatively. Twenty-four patients (mean age 68 years; range, 36 to 84 years) undergoing elective hip or knee arthroplasty underwent measurements of TcPO2 preoperatively, 2 days immediately postoperatively, and at 2-month follow-up. They reported that immediately after surgery the TcPO2 values decreased, but improved in the 2 months after surgery. This decrease in TcPO2 values were attributed to the expected changes in oxygen delivery, metabolism, and diffusion after surgery—hyperemia, edema due to inflammatory response [13,14], as well as trauma to the microvasculature of the wound site. The increase in the metabolic demand of the wound site tissue in comparison to normal tissue was also suggested. The use of TcPO2 in evaluating lower-extremity perfusion after angioplasty has been extensively reported by others. Caselli et al [15] assessed the TcPO2 values of 43 diabetic patients with ischemic foot ulcers of which 23 underwent successful revascularization via PTA. The authors evaluated TcPO2 preoperatively as well as 1, 7, 14, 21, and 28 days post procedure. After revascularization, TcPO2 progressively improved in the successfully revascularized group, with a TcPO2 >30 mm Hg in 38.5% of patients 1 week after PTA and reaching its peak value in the 4th week by increasing to 75%. These results have been confirmed by others [16,17]. Pardo et al [16] performed a prospective study with 151 patients, 64 of which underwent revascularization via PTA, and then evaluated TcPO2 pre- and postoperative and compared with ankle
brachial index (ABI). The investigators assessed the posterior tibial and dorsalis pedis arteries by duplex scanning. When two or more of these studies returned an abnormal result, angiography would then be performed. They reported that after angioplasty, the ABI significantly increased from 0.67 7 0.3 to 0.84 7 0.3, and the TcPO2 increased from 27.20 7 11.1 mm Hg to 40 7 12.1 mm Hg. Additionally, TcPO2 could be measured in all patients, and the ABI could not be measured in 25.4% pretreatment and in 17.91% post treatment. Kim et al [17] reported on 29 diabetic patients with

6

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

Table 2 – Overview of regional or targeted perfusion studies. Modality TcPO2

his

ICGA

SPECT

Laser Doppler

Description

Benefits

Limitations

Physiologic testing to evaluate potential wound healing by measuring the partial pressure of O2 in tissue Scanning spectroscopy to display tissue perfusion on a microvascular level. Measures oxyhemoglobin and deoxyhemoglobin, along with surface temperature Traditional angiography with injection of intravascular contrast agents to visualize the vasculature and areas of tissue perfusion Employ small amounts of radioactive substances that are injected into a vein and used with special cameras to produce images of the lower-extremity vasculature and angiogenesis Uses light penetration and absorption to evaluate microcirculatory perfusion

Fast, noninvasive, cost effective Office/clinic application Noninvasive Can be used for surveillance imaging post revascularization procedure Can be used to monitor perfusion closely Can perform on the spot interventions Noninvasive Can be used for surveillance imaging post revascularization procedure Fast, noninvasive, cost effective

The accepted level of TcPO2 that indicates tissue healing remains controversial. No large-scale studies have been undertaken to verify the reliability of measurements in patient with PAD Nephrotoxic contrast agents Costly and time consuming Invasive study requiring direct arterial puncture for access No large-scale studies have been undertaken to verify the reliability of measurements in patient with PAD Cannot provide absolute perfusion values, must combine with other modalities

HSI, hyperspectral imaging; ICGA, indocyanine green angiography; PAD, peripheral arterial disease; SPECT, single photon emission tomography; TcPO2, transcutaneous partial pressure of oxygen.

nonhealing ischemic ulceration of the lower extremity who also underwent PTA with serial TcPO2 measurement throughout the perioperative period up to 6 weeks after the intervention. They considered the PTA successful, acceptable, or failed when residual stenosis was o30%, between 30% and 50%, and >50%, respectively. Immediately after PTA, 26 feet were evaluated as being successful and the remaining 3 feet were acceptable. In terms of TcPO2, the mean measurement was 12.7 7 8.9 mm Hg, with subsequent improvement during the following weeks, and an eventual mean TcPO2 value of 53.8 7 21.0 mmHg by week 6. Andrews et al [18], conducted a retrospective observational study of 307 patients who underwent partial foot amputation. They performed serial noninvasive vascular studies, with the affected extremity in both dependent and elevated positions during the perioperative period, to evaluate the success or failure of TcPO2 in determining appropriate level of amputation. Their endpoint was either successful wound healing or need for re-amputation. They reported that a TcPO2 value of >38 mm Hg had a sensitivity and specificity of 71% for predicting healing or failure. Misuri et al [19] similarly evaluated 30 patients withTcPO2 measurements before undergoing amputation due to CLI to evaluate the cut-off value for wound failure or revision amputation. Transcutaneous oxygen tension was measured at the dorsum foot and on the upper third of the anteromedial calf. Of this population, 23 underwent minor amputations (transmetatarsal or digit), and 7 underwent a below-knee amputation. They found that 15 of 17 patients with successful amputations had a TcPO2 value >20 mm Hg, and 11 of 13 amputations that failed had a TcPO2 r20 mm Hg. The findings were statistically significant, with a sensitivity of 88.2% and specificity 84.6% in predicting success or failure by this modality. Although there is a correlation with decreasing TcPO2 values and increased need for re-

amputation, no study has definitively shown that this modality should be used solely in the selection of amputation sites in patients failing revascularization. Arsenault et al [20] performed a systemic review and meta-analysis to determine the validity of TcPO2 as a predictor of lower-limb amputation healing and development of complications postoperatively. A total of 31 studies were analyzed, with 1,824 patients undergoing 1,960 amputations. The review did suggest that there was an inverse relationship between decreasing TcPO2 values and increasing rate of amputation healing failure. However, they could not ascertain an appropriate cut-off value for use as a standard in clinical practice, as when TcPO2 values fell to o40 mm Hg, there was some variation in the percentages of amputation healing failure. This analysis suggests that although TcPO2 predicts healing complications of lower-limb amputations, the independent predictive value cannot be precisely determined. TcPO2 is an older noninvasive modality that has been studied for a variety of medical applications since the 1980s. Its utility in the field of vascular surgery is that it allows the clinician to closely evaluate the microcirculation and tissue perfusion in specific segments of the foot post revascularization, as well as a supplement to clinical examination in predicting the likelihood of wound failure in patients requiring amputation. Although the level of TcPO2 that predicts wound healing remains controversial, it is generally accepted that wounds are likely to heal if oxygen tension is >40 mm Hg (in the absence of diabetes, infection, and tissue edema). Patients with values of o20 mm Hg are severely ischemic and will likely require either revascularization or amputation for lower-extremity ulceration. However, as stated by Arsenault et al [20], a large sufficiently powered study that incorporates multivariable analysis is needed to further identify its use in clinical practice.

SE

4.

M I N A R S I N

V

A S C U L A R

Hyperspectral imaging

Hyperspectral imaging (HSI) is a new modality for the evaluation of PAD and CLI that utilizes scanning spectroscopy to construct spatial maps for tissue oxygenation using wavelengths (between 500 and 660 nm) of visual light (Fig. 2). By combining digital imaging with conventional spectroscopy, targeted wavelengths for the absorption peaks for oxyhemoglobin and deoxyhemoglobin can be identified and measured. These wavelengths of light penetrate to 1 to 2 mm below the skin and provide information from the subpapillary plexus. The subpapillary plexus is where the subcutaneous arteries form a network in the subcutaneous tissue and supply the skin with blood. Chin et al [21], demonstrated measurable differences in the tissue oxygenation of 222 limbs along angiosome regions of the foot in patients with and without PAD. This was a 10week prospective study evaluating the utility of detecting tissue oxygenation levels for correlation with severity of PAD. The study evaluated 111 patients with 222 limbs, who comprised 65 patients with PAD and 46 patients without PAD. The oxyhemoglobin levels in the skin below the ankle of

Fig. 2 – Top: visual imaging of both peripheral arterial disease (PAD) and non-PAD patients. Center: integrated oxyhemoglobin-deoxyhemoglobin (Oxy-Deoxy) hyperspectral imaging of both PAD and non-PAD patients. (Bottom) Deoxyhemoglobin (Deoxy) hyperspectral imaging of both PAD and non-PAD patients. The foot with PAD has substantially decreased oxyhemoglobin and deoxyhemoglobin values throughout the angiosome [21].

SU

R G E R Y

27 (2014) 3–15

7

both PAD and non-PAD did not display any difference; however, there was a significant difference in the content of deoxyhemoglobin found along the plantar angiosomes, which encompasses the plantar metatarsal, plantar arch, and plantar heel. An example of this is in Figure 2, displaying visual, integrated oxyhemoglobin-deoxyhemoglobin, and deoxyhemoglobin HSI [21]. In these angiosomes, the level of deoxyhemoglobin was found to be decreased in patients with PAD compared to non-PAD patients. This was in contrast to a previous study by Jafari-Saraf et al [22], that did not find a correlation between the oxyhemoglobin or deoxyhemoglobin in PAD patients. This discrepancy could be attributable to the researchers evaluating the dorsum and ankle of the foot, whereas Chin et al [21] evaluated nine angiosomes, including the plantar aspect. The glaborous skin of the plantar aspect maintains a rich network of arteriovenous anastomoses that would likely maintain higher oxygenation levels. It was speculated that for chronic PAD the arterial flow for both macroperipheral and microperipheral vessels maintain increased blood flow to the skin of the lower extremity when in dependent position due to decreased precapillary resistance. The mechanism underlying this phenomenon is both the impairment of the sympathetic postural autonomic vasoregulation mechanism and the venoarterial response. The angiosome regions were also concurrently evaluated with Doppler and that did demonstrate a downward trend in deoxyhemoglobin content, as the signal decreased from triphasic to biphasic, and finally monophasic. This finding is likely due to a restriction in deoxyhemoglobin flow, which suggests that increasing deoxyhemoglobin correlates with increasingly severe PAD. HSI has also been shown to identify changes in the skin microcirculation in diabetic patients as found by a study published by Nouvong et al [23]. The investigators performed a prospective multicenter 24-week study demonstrating that HSI provides a local assessment of microvascular oxygenation status that is predictive of ulcer healing in 54 patients with 73 ulcers who had either type 1 or type 2 diabetes mellitus. Ulcers were classified into one of two groups: ulcers that healed within 24 weeks or ulcers that did not heal within 24 weeks. Their primary endpoint was to establish the effectiveness of hyperspectral tissue oxygenation mapping for predicting whether diabetic foot ulcers in both type 1 and 2 diabetic patients would heal. The study revealed higher oxyhemoglobin levels in the 85% of diabetic foot ulcers that healed versus the 64% that did not heal. They concluded that HSI offers high sensitivity (86%) and specificity (88%) in determining healing potential when the researchers removed three false-positive osteomyelitis cases and four falsenegative cases due to improper measurements on calluses instead of ulceration. As an imaging modality, HSI remains in its infancy; however, its potential utility in vascular assessment is high if it can be further validated. As yet there is no definitive method to predict wound healing, and the study by Nouvong et al [23] does demonstrate the feasibility with HSI. Compared to existing tools, such as ABI/segmental pressures, HSI can deliver a much finer assessment of perfusion in specific anatomic areas that can be easily understood in its anatomic oxygenation maps in contrast to gross levels within the leg,

8

SE

M I N A R S I N

V

A S C U L A R

as in ABIs. Its noninvasive nature cannot be understated, as no patient contact is necessary to image the target area. The use of visual wavelengths of light can further protect patients from exposure to ionizing radiation. Anatomic maps can be rendered in other modalities, such as indocyanine green (ICG) angiography and single photon emission tomography (SPECT) imaging (see section titled Indocyanine Green Angiography); however, HSI avoids the use of intravenous contrast agents, which often require more highly trained personnel, elaborate examination areas, and supply storage facilities. HSI does face hurdles to accuracy. Although this has not been definitively studied, it might still remain vulnerable to weaknesses faced by other skin perfusion detectors, such as TcPO2. Inflammatory reactions, such as that induced by infection, could cloud the interpretation of measurements in with local hyperemia exists. Target area positioning will also likely need to be examined and standardized because the study by Chin et al [21] suggested detectable changes with the venoarteriolar reflex with ischemia. The long-term and large population validity of HSI no doubt requires more extensive testing, yet, as a diagnostic and prognostic tool, it certainly has important potential advantages. HSI has demonstrated an ability to show real-time perfusion of the angiosome for preoperative planning. This technology can potentially evaluate the level of reperfusion after an intervention to monitor success or failure after index procedure. Currently, there are no large-scale studies published that evaluate this application, but it would be reasonable to assume that, based on these results, HSI could be used for both the preoperative and postoperative phase of PAD treatment.

5.

Indocyanine green angiography

Digital subtraction angiography remains the gold standard of all imaging modalities for assessing the accuracy of other technologies from an anatomic perspective, as it allows the highest spatial resolution of any imaging study. Another important advantage is that it provides the opportunity to intervene on any discovered lesions using endovascular techniques. As an adjunct to digital subtraction angiography and for its use in assessment of perfusion, ICG angiography has been studied. The technique of ICG angiography uses a low-power laser coupled with a charge-coupled device camera to sequence ICG perfusion at the surface of the skin. ICG (Fig. 3) is an inert, water-soluble, nonradioactive, and relatively nontoxic contrast agent approved by the US Food and Drug Administration in 1959. ICG toxicity is low, but it does contain sodium iodide, so caution should be exercised in patients with history of iodine allergies [24]. ICG is rapidly bound to plasma albumin before undergoing hepatic metabolism, and has a relatively short half-life of 3 to 5 minutes; as such, it can be utilized safely in patients with renal insufficiency. When ICG absorbs light it fluoresces at a wavelength between 750 and 880 nm. In comparison to hemoglobin, which absorbs light at a level of 650 nm, and water, which absorbs light at >900 nm (Fig. 4), there is an optical window where the fluorescent activity of ICG could be observed and is near-infrared light range. ICG is made to fluoresce by a laser

SU

R G E R Y

27 (2014) 3–15

Fig. 3 – The structure of indocyanine green [24].

light source and camera system (which is held perpendicular to the target region), and the intensity of fluorescence is proportional to the rate of perfusion in the affected tissue. The areas of fluorescence intensity can be viewed in grayscale, with whiter imaging indicating higher intensity, or as a heat map, where red indicates high intensity and blue indicate low intensity (Fig. 5). Multiple data points can be analyzed, including the starting fluorescent intensity upon initiation of the ICG angiography study (starting intensity), the magnitude of intensity increase from baseline to peak intensity (ingress), the rate of intensity increase from baseline to peak intensity over time (ingress rate), the area under the curve of intensity over time (curve integral), the intensity at the end of the study (end intensity), the magnitude of intensity decrease from peak intensity to the end of the study (egress), and the rate of intensity decrease from peak intensity to the end of the study (egress rate). The clinical utility of ICG angiography has been explored in PAD and CLI patients for the diagnostic evaluation and planning of angiosome-based direct revascularization. Braun et al [25], performed a 16-month study of 24 patients who underwent preprocedural ICG angiography, after obtaining ABI and toe pressures before undergoing 31 revascularization procedures. They utilized the SPY system (Novadaq, Bonita Springs, FL) to perform their imaging. In 50% of the patients, ABI and toe pressures were unreliable due to medial calcinosis. Additionally, 50% of patients had one or two tibial vessels with occlusion. The patients underwent open bypass (13%), endovascular therapy (84%), or were treated with a hybrid procedure (3%). A small subset of this group (n ¼ 13), underwent additional post-procedural ICG angiography to evaluate perfusion in the affected limb. In the follow-up period, 23% of patients had complete healing of their lowerextremity ulcers in a 6-month time frame. Two of those patients had undergone repeat revascularization due to developing contralateral CLI. In patients who underwent repeat ICG angiography (Fig. 5), comparison could be made showing the increasing perfusion of the affected angiosome after targeted intervention. A case report by Perry et al [26] utilized ICG angiography to clarify the extent of necessary debridement and provided an immediate indication of improvement in regional perfusion status after revascularization. A recent abstract from Braun et al [27], details a study of 46 patients from January 2011 to December 2013 that underwent 57 revascularizations for CLI and tissue loss. The patients had ABI taken while undergoing ICG angiography both preoperatively and (within 5 days) postoperatively to evaluate perfusion. Sixty-five percent of patients had incompressible ABI before intervention. After revascularization and subsequent ICG angiography, there was a statistically

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

9

Fig. 4 – Excitation and emission spectra of indocyanine green: excitation wavelength (yellow line), emission wavelength (red line), hemoglobin (pink line), and water (black line) absorbance spectra [24]. significant correlation (P o .05) between ABI and degree of ingress, as well as ingress rate for both pre and post interventions. Eighty percent of those patients with a previous ingress of 27 pixels, as well as an ABI of 0.4, were found post revascularization to have >27 pixels and a >ABI of 0.4, with 85% having ingress rates >1.1 pixels/s and 100% of those patients with compressible ABI postoperatively were >0.4. The parameters of ingress as well as ingress rate appeared to correlate with improved perfusion after revascularization,

and are objective data that are quantifiable and easily obtained when evaluating perfusion. Terasaki et al [28], evaluated the use of ICG angiography as an adjunct to distal pressure measurements in patients with symptomatic PAD and CLI, utilizing the Photodynamic Eye System (Hamamatsu K.K. Hamamatsu, Japan). They performed ICG angiography as part of the preoperative workup, with administration of ICG via the brachial vein while performing continuous ICG angiography for 5 minutes. There

Fig. 5 – (A) An indocyanine green angiography (ICGA) grayscale image with curve is shown in the patient pre-intervention. (B) An ICGA grayscale image with curve for the patient post-intervention shows a sixfold increase in the ingress rate and appearance of egress noted on bottom of image. ICGA heat-map imaging is shown for both (C) pre-intervention and (D) postintervention, with the post-intervention values recorded as elevated by the green labels [25].

10

SE

M I N A R S I N

V

A S C U L A R

was a 20-second delay before fluorescent activity was visualized on the scanner and findings recorded. The intensity of fluorescence was then plotted on a time-intensity curve with the severity of ischemia defined as the duration between the rising point and half the value of maximum brightness (T1/2). There was a comparison of fluorescent intensity at the 10-second mark (PDE10) with TcPO2 at those sites to evaluate for possible correlation in CLI. They evaluated total of 34 patients, 16 with ulceration or tissue loss (Fontaine class IV), 11 with claudication (Fontaine class II), and 7 with rest pain (Fontaine class III). They found that the median T1/2 in Fontaine II patients was 23 seconds, Fontaine III was 41 seconds, and Fontaine IV was 17 seconds. The issues with median T1/2 as an objective parameter is that conditions within the foot could false elevate the value, such as inflammation and hemodynamic status, which, if low, could provide a falsely low value. Other factors, such as body habitus and penetration of light into target tissue, can further skew the value of T1/2. The best correlation was demonstrated with PDE10 and TcPO2: in Fontaine class IV patients with PDE10 value of 28 (calculated from ROC curve) was used to identify tissues with TcPO2 o30 mm Hg. The calculated sensitivity and specificity were 100% and 86.6%, respectively. Igari et al [29,30] evaluated the use of ICG angiography during digital subtraction angiography in patients with PAD and CLI, pre and post revascularization. The foot was divided into regions of interest (ROI) (Fig. 6): (1) from the Chopart joint to the Lisfranc joint; (2) at the metatarsal bones; and (3) in the distal region of the first metatarsal bone. The patients affected limb had Rutherford classification from 2 to 4, with the majority in class 3. The equipment used was the same as detailed by Terasaki et al [28]. The patients underwent revascularization via an open, hybrid, or endovascular technique with both pre- and postoperative ICG angiography to evaluate perfusion. The parameters recorded from ICG angiography (Fig. 7) included the magnitude of intensity from ICG onset to maximum intensity (Imax), the time from ICG onset to maximum intensity (Tmax), the slope of the intensity increase from ICG onset to maximum intensity (S), the time elapsed from the fluorescence onset to half the maximum intensity (T1/2), and the fluorescence intensity measured 10 seconds after the onset of fluorescence (PDE10). With respect to the measurements of ROI 1, 2, and 3, the values of Imax, Tmax, S, T1/2, and PDE10 were all significantly different between the pre- and postinterventional ICG angiography tests. ICG angiography parameters were then compared to traditional parameters in noninvasive monitoring. The ABI exhibited a statistically significant correlation with the Tmax, S, T1/2, and PDE10 in ROI 1, and the Imax, Tmax, S, T1/2, and PDE10 demonstrated significant correlations with each other in ROI 2, and ROI 3. However, as noted in previous studies, the intensity of ICG does depend on the distance from the camera to the skin, the patient's skin color, and the ambient light in the testing room, as reported in a prior studies. Thus, the intensity of ICG may not be a good parameter for assessing tissue perfusion using ICG angiography. Instead, the study indicates that parameters based on the time after ICG injection may be the best markers of perfusion. Because the prognosis of ischemic vascular disease is directly related to the functional perfusion level, rather than

SU

R G E R Y

27 (2014) 3–15

Fig. 6 – Region of Interest (ROI) 1 ¼ Chopart joint to lisfrainc joint, ROI 2 ¼ metatarsal bones, and ROI 3 ¼ distal region of the first metatarsal [29]. merely a vascular structure, functional perfusion imaging is superior to structural vascular imaging in guiding targeted therapy via the angiosome model. Thus, ICG angiography has been a focus of interest because of its convenience and effectiveness for imaging the vasculature. The test allows a quantitative estimation of tissue perfusion and real-time

Fig. 7 – X-axis is time measured in seconds. Y-axis is represents the intensity of fluorescence. (A) Imax is the magnitude of intensity from ICGA onset to maximum intensity; (B) Tmax is the time from ICGA onset to maximum intensity; (C) S ¼ Imax/Tmax is the slope of the intensity increase from ICG onset to maximum intensity; (D) T1/2 is the time elapsed from the fluorescence onset to half the maximum intensity; (E) PDE10 is the fluorescence intensity measured 10 seconds after the onset of fluorescence [29].

SE

M I N A R S I N

V

A S C U L A R

assessment of perfusion after a revascularization procedure. In addition, ICG angiography tests are useful as minimally invasive tools for determining the tissue viability in patients who lack toe pulses due to ulceration or amputation of the toes, or patients with an abnormal ABI due to medial calcification. Further research must be done to establish a standard technique, as there was variation between equipment and practices in each of the studies mentioned. A comparison between intensity of signal and initial detection of fluorescence has to be performed to determine which parameter most accurately depicts perfusion in the lower extremity.

6.

Nuclear diagnostic imaging

Nuclear imagers have been using radioisotopes for years to assess myocardial perfusion, however, only recently have these same clinical tools been translated into assessing perfusion in patients with PAD. Recent advancements in the field of positron emission tomography (PET) and SPECT technology have allowed for the targeting and imaging of more specific cellular processes. SPECT/PET imaging can visualize perfusion and the process of angiogenesis in affected ischemic tissue by providing a combination of high-sensitivity radiotracer-based imaging with highresolution CT scan imaging obtaining both functional and structural information to more effectively evaluate the disease process and supplementing clinical judgment. SPECT followed by CT scanning provides clinicians a noninvasive tool to determine areas of high and low tracer uptake. The tracers are general perfusion markers, such as Myoview (99mTc); commercial radiolabeled perfusion molecules; or can be specifically labeled to only target certain membrane peptides or areas of low pH. Using perfusion markers on patients with PAD or CLI potentially allows clinicians to assess changes in perfusion along the length of the lower extremities without an invasive procedure such as angiography. In addition, nuclear technology is the only diagnostic tools that offer clinicians the ability to look at the lower extremity in a three-dimensional manner. SPECT scans of the lower extremities using technetium perfusion tracers can be used before and after intervention to help assess the degree of tissue perfusion and determine if the intervention was successful (Fig. 8) [31,32]. By applying the angiosome model to SPECT data analysis, it provides vascular specialists with a quick and effective way to determine the effect of the revascularization. The advantage of this testing over the current clinical tests is its focus on tissue blood perfusion. If the clinicians are primarily interested in increasing blood flow to the area of an ulcer, SPECT scanning can tell them instantly if the intervention actually increased blood flow to that area (Fig. 8). Nuclear imaging tracers have advanced beyond just simple perfusion makers, to now being able to be localized to molecules, proteins, and even fats. Angiogenesis, or growth of new capillaries from microvessels, occurs as a result of ischemic injury, hypoxia, inflammation, shear stress, or traumatic injury. Imaging of angiogenesis can be divided into targeting three major cell groups: (1) nonendothelial cell types (stem cells, macrophages, and monocyte); (2) endothelial cell

SU

R G E R Y

27 (2014) 3–15

11

targets (vascular endothelial growth factor [VEGF], growth factor receptors, and cell adhesion molecules); and (3) extracellular matrix proteins and proteases. Although many angiogenesis-stimulating factors exist, VEGF is considered the most potent and predominant factor in the stimulation of angiogenesis, so research led to the development of multiple molecular radiotracer probes (Table 3), which are labeled with 124I or 123I for VEGF receptors for myocardial ischemia and PAD, which showed up-regulation during angiogenesis. There have been many studies [31] that have focused on targeted imaging of angiogenesis in animal models of induced ischemia. VEGF121 labeled with 111In has been developed as a targeting ligand for SPECT and was successfully used to image peripheral angiogenesis in a rabbit model of hind limb ischemia in the study by Lu et al [33]. Similarly, Willman et al [34] performed a study based on the murine model of hind-limb ischemia-induced angiogenesis has revealed that 64Cu6DOTA-VEGF121 is effective in PET imaging of VEGFR-2. Another target for molecular probes to identify angiogenesis is αvβ3 integrin [35], which is found in abundance on the surface of proliferating endothelial cells and is specific marker of ongoing angiogenesis. Tc-NC100692 (99m Tc-maraciclatides) is a technetium-radiotracer that has been used in a variety of SPECT [36] studies to noninvasively assess angiogenesis, as it has high affinity for the αvβ3 integrin, is both metabolically stable and has a biodistribution that is favorable for SPECT imaging (Fig. 9). Nuclear imaging offers clinicians a new and improved way to detect and assess patients with PAD. However, these advancements come at a large cost. SPECT machines are not only extremely expensive, costing upward of a couple million dollars; they also require a team to trained nuclear technicians to run each scan. In addition, hospitals need a way to produce the radioactive isotopes everyday, because many of them have short half-lives: 6 hours for SPECT and around 30 minutes for PET. Although many large hospital systems already have nuclear laboratories used for cardiac imaging, smaller hospitals may not afford these machines.

7.

Laser Doppler

Laser Doppler, initially described by Stern et al [37], functions by measuring the total local microcirculatory blood perfusion, including the perfusion in capillaries, arterioles, venules, and shunting vessels. The device emits a beam of laser light that is then scattered and partially absorbed as it penetrates the tissue being evaluated. Within the tissues, light will strike a moving object, such as blood cells, which then creates a change in its wavelength known as the Doppler shift. Lights hitting any static objects will remain unchanged. The magnitude and frequency distribution of these changes in the wavelength are related to the concentration of blood cells in tissue or the tissue perfusion in the targeted region. This fraction of light that is then detected by a remote photodiode is then converted into an electrical signal (Fig. 10). In the processor, a signal proportional to the tissue perfusion at each measurement point is calculated and stored. No current laser Doppler can provide absolute perfusion values (eg, mL/ min/100 g tissue); instead measurements are expressed as

12

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

Fig. 8 – Multimodality evaluation with (A) ankle-brachial indices, (B) computed tomography (CT) angiography, and with a (C) hybrid 99mTc-tetrofosmin single photon emission tomography (SPECT)/CT reveals impaired lower-extremity pressures and tissue perfusion in peripheral arterial disease patient with previously implanted aortoiliac stents. (D) Segmentation of muscle groups (Red 5 gastrocnemius; yellow ¼ soleus; green ¼ tibialis; and purple ¼ fibularis) into 3-dimensional regions of interest by CT attenuation images (E) confirmed differences in regional tissue perfusion between legs. ABI ¼ ankle-brachial index; PPG ¼ photoplethysmograph; PVR ¼ pulse volume recording; TBI ¼ toe-brachial index [32].

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

13

27 (2014) 3–15

Table 3 – A summary of molecular probes used for noninvasive imaging of angiogenesis [31]. Marker VEGF VEGF VEGF VEGF VEGF VEGF VEGF αvβ3 αvβ3 αvβ3

Probe 124

I-VG76e I-VEGF165 111 In-VEGF121 64 Cu-VEGF121 64 Cu-VEGF121 99m Tc-scVEGF 64 Cu-scVEGF 111 In-RP748 111 In-RP748 18 F-AH111585 123

Imaging modality

Biologic target

PET SPECT SPECT PET PET SPECT PET SPECT SPECT PET

Tumor angiogenesis Tumor angiogenesis Peripheral limb angiogenesis Tumor angiogenesis Myocardial angiogenesis Peripheral limb angiogenesis Tumor angiogenesis Tumor angiogenesis Myocardial angiogenesis Tumor angiogenesis

PET, positron emission tomography; SPECT, single photon emission tomography VEGF, vascular endothelial growth factor.

relative perfusion units. When the scanning procedure is completed, the system generates a color-coded perfusion image on a monitor [38]. Laser Doppler has been shown to identify poor perfusion in lower extremities ulcers, Ambrozy et al [38], used laser Doppler perfusion imaging and capillary microscopy for assessing both subpapillary and nutritive microcirculation in four defined regions of the skin in 17 patients with mixed ulcers caused by a combination of PAD and chronic venous insufficiency. For each ulcer, a nongranulation tissue area, a granulation tissue area, and adjacent skin area were defined. In these areas, the average laser Doppler area flux (arbitrary units) and the number of capillaries/mm2 were determined for each patient. In the nongranulation tissue area, low laser Doppler area flux is combined with very low capillary density (ulcer area without healing). In granulation tissue area, the highest laser Doppler area flux of all three areas and an intermediate capillary density (wound healing) is measured. In skin area, an intermediate laser Doppler area flux is asso-

ciated with the highest capillary density of all three areas with the healing process nearly completed and no granulation tissue. Ludyga et al [39], evaluated the ABI in patients with PAD including symptomatic CLI, compared the ABI obtained with traditional Doppler and sphyngomamometer versus laser Doppler. The 30 patients, 21 men and 9 women, underwent the treadmill test, and pain-free walking distances were measured with both Doppler ultrasound (ABI-Doppler) and laser Doppler (ABI-laser Doppler). There was a comparable correlation between both ABI-Doppler and ABI-laser Doppler with relation to claudication distance. Although there was a slightly higher correlation with measurements of claudication distances taken by ABI-Laser Doppler. This could reflect the fact that laser Doppler allows for the evaluation of both micro- and macrovascular circulation. Laser Doppler is also not limited by the technical skill of the user, is faster and simpler, although increased unit costs limits its use in the clinical field.

Fig. 9 – Positron emission tomography/computed tomography imaging of angiogenesis in a murine model of hind limb ischemia. (A) Nontargeted dendritic nanoprobes (bottom center). (B) Higher uptake of αvβ3-targeted dendritic nanoprobes in ischemic hind limb (left side of image) than in control hind limb (right side of image) [35].

14

SE

M I N A R S I N

V

A S C U L A R

SU

R G E R Y

27 (2014) 3–15

Fig. 10 – The device emits a beam of laser light that is then scattered as it penetrates the tissue while being partially absorbed in the tissue being evaluated. Light will strike a moving object, such as blood cells, within both arteries and veins to create a change in its wavelength, known as the Doppler shift. This fraction of light that is then detected by a remote photodiode is then converted into an electrical signal [37]. In the study by Ray et al [40], 41 patients with symptomatic PAD and CLI underwent 30 revascularizations during a 6month course had both TcPO2 and laser Doppler fluxmetery (LDF) measured both pre- and postoperatively. In healthy subjects, peak LDF is usually observed 20 to 30 seconds after restoration of flow, while in claudicants, peak LDF may be delayed for >60 seconds. LDF at the toe is normally reduced by between 30% and 50% when supine healthy subjects assume the sitting position, LDF in a leg threatened by severe ischemia increases by as much as threefold during leg dependency. Peak LDF is preferred over resting LDF, as there is greater variability in the latter’s reproducibility. It has been identified in previous studies that the time to peak LDF after a period of ischemic occlusion (2 minutes in this study,) is closely related to total limb vascular resistance, as well as vascular ischemia. A peak LDF in excess of 100 seconds correlated with increased risk of failing revascularization or amputation wound healing.

8.

Summary

Modern practice in revascularization for CLI patients still revolves around traditional “best artery” for bypass or angioplasty. However, several contemporary revascularization series report that the rate of unhealed ischemic wounds, despite intervention, remains at up to 18% of cases resulting in amputation [5,6]. Current research suggests that the angiosome model or targeted approach may provide substantially better results for revascularization and limb salvage [8]. With the knowledge that the prognoses for PAD and CLI are closely correlated with the functional perfusion level of the affected extremity rather than the macrovascular structure, regional foot perfusion imaging may predict wound healing success and function as a dependable surveillance tool. The clinical evaluation of the angiosome model could only be truly realized if a proper imaging system is in place that is noninvasive, fast, and safe, and can easily delineate wound topography to guide directed

revascularization therapy. Some of these modalities, such as SPECT/PET, have wide and varied future applications, like delivery of targeted drug therapy using nanoprobes. Many of theses imaging systems are in the infancy; their clinical application would require additional long-term and large population trials to ensure efficacy and to develop future protocols. With increasing interest and continued refinement in our understanding of PAD/CLI, the field of vascular surgery moves toward achieving a significant reduction in persistent ulceration and decreasing the rate of complications after revascularization for our patients. To accomplish this, we must be willing to adapt new paradigms and techniques in the treatment of this complex disease process. The implementation of these newer modalities as part of our routine clinical evaluation appears increasingly closer as each individual technology is optimized and we understand how to better utilize them effectively in conjunction with clinical judgment.

re fe r en ces

[1] Berceli SA, Chan AK, Pomposelli FB Jr, et al. Efficacy of dorsal pedal artery bypass in limb salvage for ischemic heel ulcers. J Vasc Surg 1999;30:499–508. [2] Dorros G, Jaff MR, Dorros AM, et al. Tibioperoneal (outflow lesion) angioplasty can be used as primary treatment in 235 patients with critical limb ischemia: five-year follow-up. Circulation 2001;104:2057–62. [3] Taylor GI, Palmer JH. The vascular territories (angiosomes) of the body: experimental study and clinical applications. Br J Plast Surg 1987;40:113–41. [4] Taylor GI, Pan WR. Angiosomes of the leg: anatomic study and clinical implications. Plast Reconstr Surg 1998;102: 599–616, discussion 617
8. [5] Attinger CE, Evans KK, Bulan E, et al. Angiosomes of the foot and ankle and clinical implications for limb salvage: reconstruction, incisions, and revascularization. Plast Reconstr Surg 2006;117(suppl):261S–293SS. [6] Clemens MW, Attinger CE. Angiosomes and wound care in the diabetic foot. Foot Ankle Clin 2010;15:439–64.

SE

M I N A R S I N

V

A S C U L A R

[7] Inoue Y, Taylor GI. The angiosomes of the forearm: anatomic study and clinical implications. Plast Reconstr Surg 1996;98:195–210. [8] Sumpio BE, Forsythe RO, Ziegler KR, et al. Clinical implications of the angiosome model in peripheral vascular disease. J Vasc Surg 2013;58:814–26. [9] Söderström M, Albäck A, Biancari F, et al. Angiosometargeted infrapopliteal endovascular revascularization for treatment of diabetic foot ulcers. J Vasc Surg 2013;57: 427–35. [10] Iida O, Soga Y, Hirano K, et al. Long-term results of direct and indirect endovascular revascularization based on the angiosome concept in patients with critical limb ischemia presenting with isolated below-the-knee lesions. J Vasc Surg 2012;55:363
70 e5. [11] White RA, Nolan L, Harley D, et al. Noninvasive evaluation of peripheral vascular disease using transcutaneous oxygen tension. Am J Surg 1982;144:68–75. [12] McPhail IR, Cooper LT, Hodge DO, et al. Transcutaneous partial pressure of oxygen after surgical wounds. Vasc Med 2004;9:125–7. [13] Dooley J, Schrimer J, Slade B, et al. Use of transcutaneous pressure of oxygen in the evaluation of edematous wounds. Undersea Hyperb Med 1996;23:167–74. [14] Boyko EJ, Ahroni JH, Stensel VL, et al. Predictors of transcutaneous oxygen tension in the lower limbs of diabetic subjects. Diabet Med 1996;13549
54 1996;13. [15] Caselli A, Latini V, Lapenna A, et al. Transcutaneous oxygen tension monitoring after successful revascularization in diabetic patients with ischaemic foot ulcers. Diabet Med 2005;22: 460–5. [16] Pardo M, Alcaraz M, Ramón Breijo F, et al. Increased transcutaneous oxygen pressure is an indicator of revascularization after peripheral transluminal angioplasty. Acta Radiol 2010;51:990–3. [17] Kim HR, Han SK, Rha SW, et al. Effect of percutaneous transluminal angioplasty on tissue oxygenation in ischemic diabetic feet. Wound Repair Regen 2011;19:19–24. [18] Andrews KL, Dib MY, Shives TC, et al. Noninvasive arterial studies including transcutaneous oxygen pressure measurements with the limbs elevated or dependent to predict healing after partial foot amputation. Am J Phys Med Rehabil 2013;92:385–92. [19] Misuri A, Lucertini G, Nanni A, et al. Predictive value of transcutaneous oximetry for selection of the amputation level. J Cardiovasc Surg (Torino) 2000;41:83–7. [20] Arsenault KA, Al-Otaibi A, Devereaux PJ, et al. The use of transcutaneous oximetry to predict healing complications of lower limb amputations: a systematic review and metaanalysis. Eur J Vasc Endovasc Surg 2012;43:329–36. [21] Chin JA, Wang WC, Kibbe MR. Evaluation of hyperspectral technology for assessing the presence and severity of peripheral artery disease. J Vasc Surg 2011;54:1679–88. [22] Jafari-Saraf L, Gordon IL. Hyperspectral imaging and ankle: brachial indices in peripheral arterial disease. Ann Vasc Surg 2010;24:741–6. [23] Nouvong A, Hoogwerf B, Mohler E, et al. Evaluation of diabetic foot ulcer healing with hyperspectral imaging of oxyhemoglobin and deoxyhemoglobin. Diabetes Care 2009;32:2056–61.

SU

R G E R Y

27 (2014) 3–15

15

[24] Miwa M, et al. The principle of ICG fluorescence method. Open Surg Oncol J 2010;226
2 2010;2. [25] Braun JD, Trinidad-Hernandez M, Perry D, et al. Early quantitative evaluation of indocyanine green angiography in patients with critical limb ischemia. J Vasc Surg 2013;57: 1213–8. [26] Perry D, Bharara M, Armstrong DG, et al. Intraoperative fluorescence vascular angiography: during tibial bypass. J Diabetes Sci Technol 2012;6:204–8. [27] Braun JD, Rajguru P, Armstrong DG, et al. Indocyanine green angiographic criteria using ingress and ingress rate to detect SVS lower extremity threatened limb classification (WIfI) grade 3 ischemia. J Vasc Surg 2014;60:538. [28] Terasaki H, Inoue Y, Sugano N, et al. A quantitative method for evaluating local perfusion using indocyanine green fluorescence imaging. Ann Vasc Surg 2013;27:1154–61. [29] Igari K, Kudo, Toyofuku T, et al. Quantitative evaluation of the outcomes of revascularization procedures for peripheral arterial disease using indocyanine green angiography. Eur J Vasc Endovasc Surg 2013;46:460–5. [30] Igari K, Kudo T, Uchiyama H, et al. Intraarterial injection of indocyanine green for evaluation of peripheral blood circulation in patients with peripheral arterial disease. Ann Vasc Surg 2014;28:1280–5. [31] Stacy MR, Maxfield MW, Sinusas AJ. Targeted molecular imaging of angiogenesis in PET and SPECT: a review. Yale J Biol Med 2012;85:75–86. [32] Stacy MR, Zhou W, Sinusas AJ. Radiotracer imaging of peripheral vascular disease. J Nucl Med 2013;54:2104–10. [33] Lu E, Wagner WR, Schellenberger U, et al. Targeted in vivo labeling of receptors for vascular endothelial growth factor: approach to identification of ischemic tissue. Circulation 2003;108:97–103. [34] Willmann JK, Chen K, Wang H, et al. Monitoring of the biological response to murine hindlimb ischemia with 64Cu-labeled vascular endothelial growth factor-121 positron emission tomography. Circulation 2008;117:915–22. [35] Almutairi A, Rossin R, Shokeen M, et al. Biodegradable dendritic positron-emitting nanoprobes for the noninvasive imaging of angiogenesis. Proc Natl Acad Sci U S A 2009;106: 685–90. [36] Morrison AR, Sinusas AJ. Advances in radionuclide molecular imaging in myocardial biology. J Nucl Cardiol 2010;17:116–34. [37] Stern MD. In vivo evaluation of microcirculation by coherent light scattering. Nature 1975;254(5495):56–8. [38] Ambrozy E, Waczulikova I, Willfort-Ehringer A, et al. Microcirculation in mixed arterial/venous ulcers and the surrounding skin: clinical study using a laser Doppler perfusion imager and capillary microscopy. Wound Repair Regen 2009;17:19–24. [39] Ludyga T, Kuczmik WB, Kazibudzki M, et al. Ankle-brachial pressure index estimated by laser Doppler in patients suffering from peripheral arterial obstructive disease. Ann Vasc Surg 2007;21:452–7. [40] Ray SA, Buckenham TM, Belli AM, et al. The predictive value of laser Doppler fluxmetry and transcutaneous oximetry for clinical outcome in patients undergoing revascularisation for severe leg ischaemia. Eur J Vasc Endovasc Surg 1997;13: 54–9.