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The Importance of Engraftment in Flap Revascularization: Confirmation by Laser Speckle Perfusion Imaging
Journal of Surgical Research 164, e201–e212 (2010) doi:10.1016/j.jss.2010.07.059
The Importance of Engraftment in Flap Revascularization: Confirmation by Laser Speckle Perfusion Imaging Paul G. McGuire, Ph.D.,* and Thomas R. Howdieshell, M.D.†,1 *Department of Cell Biology and Physiology; and †Department of Surgery, University of New Mexico HSC, Albuquerque, New Mexico Submitted for publication June 20, 2010
Background. The delivery of proangiogenic agents in clinical trials of wound healing has produced equivocal results, the lack of real-time assessment of vascular growth is a major weakness in monitoring the efficacy of therapeutic angiogenesis, and surgical solutions fall short in addressing the deficiency in microvascular blood supply to ischemic wounds. Therefore, elucidation of the mechanisms involved in ischemiainduced blood vessel growth has potential diagnostic and therapeutic implications in wound healing. Materials and Methods. Three surgical models of wound ischemia, a cranial-based myocutaneous flap, an identical flap with underlying silicone sheeting to prevent engraftment, and a complete incisional flap without circulation were created on C57BL6 transgenic mice. Laser speckle contrast imaging was utilized to study the pattern of ischemia and return of revascularization. Simultaneous analysis of wound histology and microvascular density provided correlation of wound perfusion and morphology. Results. Creation of the peninsular-shaped flap produced a gradient of ischemia. Laser speckle contrast imaging accurately predicted the spatial and temporal pattern of ischemia, the return of functional revascularization, and the importance of engraftment in distal flap perfusion and survival. Histologic analysis demonstrated engraftment resulted in flap revascularization by new blood vessel growth from the recipient bed and dilatation of pre-existing flap vasculature. Conclusions. Further research utilizing this model of graded wound ischemia and the technology of laser speckle perfusion imaging will allow monitoring of the real-time restitution of blood flow for correlation with molecular biomarkers of revascularization in an
1 To whom correspondence and reprint requests should be addressed at Department of Surgery, MSC10-5610, University of New Mexico HSC, Albuquerque, NM 87131. E-mail: thowdieshell@salud. unm.edu.
attempt to gain further understanding of wound microvascular biology. Ó 2010 Elsevier Inc. All rights reserved. Key Words: wound healing; ischemia; laser speckle perfusion imaging; revascularization; arteriogenesis; angiogenesis. INTRODUCTION
One of the most potent stimuli for new blood vessel growth or neovascularization is ischemia, which induces capillary growth to restore adequate oxygen delivery to hypoxic tissues. Ischemia initiates a number of angiogenic processes, including the release of cytokines and growth factors, the up-regulation of extracellular proteinases and cell surface molecules, and the proliferation of mature endothelial cells [1]. Avascular tissue-engineered skin equivalents have been available for years, and are used to treat wounds due to burns, trauma, surgical excision, nonhealing diabetic ulcers, and blistering skin diseases [2]. Although these products may improve wound healing, long term engraftment has not been demonstrated. It is likely that inadequate perfusion in the post-transplantation period is responsible for lack of engraftment. Whereas autologous split-thickness skin grafts with an inherent vasculature purportedly become perfused in a matter of days by direct anastomosis or inosculation of preexisting graft vessels with those of the recipient, avascular skin equivalents must become perfused entirely by neovascularization from the recipient wound bed [3]. Therefore, knowledge of the basic mechanisms involved in cutaneous revascularization is imperative for development of potential treatments which promote successful wound healing. There is avid interest in developing techniques to promote neovascularization in ischemic diseases such as peripheral arterial occlusive disease, coronary artery
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disease, and cerebrovascular disease [4]. Unfortunately, the delivery of proangiogenic agents in ongoing clinical trials has produced equivocal results, the lack of real-time assessment of vascular growth is a major weakness in monitoring the efficacy of therapeutic angiogenesis, and surgical solutions fall short in addressing the deficiency in microvascular blood supply of ischemic wounds [5, 6]. The recent commercial release (2007) of a new device, the Full-Field Laser Perfusion Imager (FLPI) from Moor Instruments, Essex, UK, offers a noninvasive and noncontact method of mapping flow fields such as capillary blood flow based on the principle of laser scatter contrast imaging. The technique works by illuminating an area of tissue with laser light to produce a high contrast random interference effect know as a speckle pattern [7]. Compared with laser Doppler imaging, the effective sampling depth of laser speckle imaging is less than 1 mm, and the resulting image is of blood flow in the microvessels of the surface layers of the tissue being sampled, for example the skin. The Moor FLPI system provides video frame rate images of microvascular flow, up to 25 images per second. Therefore, it is ideally suited to any application in which dynamic changes are occurring, such as during the cardiac or respiratory cycle, and conventional laser Doppler imaging cannot provide data with sufficient time resolution [8]. In the present study, we evaluated laser speckle contrast imaging as a method to predict the spatial and temporal pattern of wound ischemia and return of functional revascularization with corroboration by simultaneous histopathologic analysis and quantitative determination of microvascular density by CD-31 immunostaining. MATERIALS AND METHODS Mouse Myocutaneous Flap Model All animals were treated humanely in accordance with the National Research Council’s ‘‘Guide for the care and use of laboratory animals’’ as part of a protocol approved by the University of New Mexico’s animal review committee. Three surgical models of wound healing were created: a cranial-based myocutaneous flap, an identical flap with underlying silicone sheeting, and a complete incisional flap (Fig. 1). Female C57BL6 transgenic green fluorescent protein (GFP) mice, ages 4 to 6 mo, underwent anesthesia with isoflurane (1%–3%) via nose cone inhalation. Post-surgical analgesia was provided using a single subcutaneous dose (0.01 mg/kg) of buprenorphine hydrochloride. After maintenance of inhalation anesthesia, hair was removed from the mouse back skin using electric clippers, and the site was prepped with povidone-iodine and 70% ethanol. The first group of mice (n ¼ 6) underwent surgical creation of a peninsular flap (3 cm in length and 1.5 cm in width) consisting of skin, adipose tissue, and panniculus carnosus muscle by making three soft tissue incisions. The flap was elevated cranially and re-approximated to the back skin with 6-0 monofilament sutures. The silicone group of mice (n ¼ 6) underwent insertion of a 0.005 inch-thick silicone sheet (BioPlexus Corporation, Saticoy, CA) beneath the flap to separate the flap from the underlying
tissue bed to prevent engraftment. Finally, in a third group of mice (n ¼ 6), a complete incisional flap was created by making a fourth and transverse soft tissue incision resulting in complete separation of the flap from the underlying tissue prior to suture reapproximation. After surgery, each animal was singly housed and received water and food ad libitum.
Fluorescein Isothiocyanate (FITC)-Dextran Perfusion Mice were anesthetized with isoflurane and were perfused via the left ventricle with 30 mL of warm phosphate buffered saline (PBS) followed by 30 mL of 10% formalin containing 20 mg/mL FITC-dextran (Sigma Chemical, St. Louis, MO). Following perfusion, the back skin containing the flap was excised, placed dermal-side up, and examined using a fluorescence dissecting microscope (Leica MZ FLIII, Wetzlar, Germany).
Laser Speckle Perfusion Imaging On days 0, 1, 3, 5, 7, 10, and 14 after surgery, each animal underwent inhalation anesthesia in prone position, and laser speckle perfusion imaging was performed with the FLPI (Full-Field Laser Perfusion Imager; Moor Instruments, Essex, UK) in low resolution/ high speed setting at a display rate of 25 Hz, time constant of 1.0 s, and camera exposure time of 20 ms. The instrument head containing the CCD (charged coupled device) camera was positioned 30 cm above the mouse back tissue surface using an articulating arm. Real-time data was acquired in the live image measurement mode. The contrast images were processed to produce a scaled color-coded live flux image (red equaled high perfusion, blue equaled low perfusion), which correlated with the blood flow velocity in the tissue. The FLPI instrument reports perfusion in arbitrary units (PU). To assign values to a measurement, the imager was calibrated using a reference flux signal which was generated by the laser light scattered from a suspension of polystyrene microspheres in water undergoing thermal or Brownian motion. From kinetic theory, the average velocity of the microspheres is proportional to the square root of the temperature in Kelvin. All measurements were performed at a room temperature of 20 C (293 Kelvin) [9]. For each time point examined, 10 single frame images acquired at end expiration (no torso movement) were analyzed in the repeat image measurement window utilizing three identical regions of interest (ROI): caudal, central, and cranial. At each time point, the mean perfusion in each ROI was calculated for control skin and deep muscle, the peninsular flap with and without silicone sheeting, and the complete incisional flap.
Tissue Harvest, Histology, GFP Intensity Expression, and Immunochemical Staining Mice were sacrificed by CO2 inhalation on days 0 (control), 3, 7, and 14 for flap harvest and histologic examination. The entire flap was excised and sectioned in transverse fashion to directly correspond to the caudal, central, and cranial laser image ROIs. The tissue was fixed in IHC Zinc Fixative (BD Biosciences-Pharmingen, San Diego, CA) for 24 h, processed, and paraffin embedded. Serial sections (4 mm) were dewaxed in xylene, taken through graded ethanol, and then hydrated in PBS solution for fluorescent and brightfield microscopy. Caudal, central, and cranial sections were mounted using ProLong Gold Antifade Reagent with DAPI (Invitrogen-Molecular Probes, Eugene, OR) for fluorescent examination. DAPI (4’,6-diamino-2phenylindole) nuclear staining provided definition for tissue orientation. Sections were examined and photographed using two-channel fluorescent microscopy at 1003 magnification (Axioskop; Carl Zeiss Microimaging, Thornwood, NY). Captured images (475 nm excitation, 509 nm emission) were analyzed for GFP intensity expression using SlideBook digital microscopy software (SlideBook 5.0, Intelligent Imaging Innovations, Santa Monica, CA). A two-dimension image mask was created, the panniculus carnosus muscle was isolated and
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FIG. 1. Proposed surgical models. (A) The peninsular-shaped, myocutaneous flap. (B) The identical flap with underlying silicone sheeting to prevent interaction of the flap with the deep fascia and back musculature. (C) The complete incisional flap without circulation. highlighted using the paint brush feature, and the software was utilized to calculate the fluorescent emission intensity. Blood vessel identity was determined by CD-31 immunostaining. Following dewaxing and hydration, sections were incubated for 10 min in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, washed in PBS, and incubated with primary antibody (rat anti-mouse CD-31, 1:50, BD Biosciences-Pharmingen) for 1 h at room temperature in a humidified chamber. Next, the sections were incubated with a biotinylated secondary antibody (anti-rat Ig HRP kit, 1:50, BD Bioscience-Pharmingen) for 30 min at room temperature. The streptavidin-HRP reagent was applied for 30 min followed by the DAB chromogen for 5 min. The sections were counterstained with Vector Hematoxylin QS (Vector Laboratories, Burlingame, CA) with quick immersion. The 3,3’-diaminobenzidine (DAB) substrate-chromogen resulted in a brown-colored precipitate at the antigen site.
Microvascular Density Determination Skin, subcutaneous tissue, and panniculus muscle vessel count and vascular luminal cross-sectional surface areas were determined with image analysis of CD-31 immunostained sections. Multiple, consistent full thickness flap biopsies were analyzed for each caudal, central, and cranial section with mean values reported. A Zeiss microscope with attached digital camera was used for image acquisition. The magnified image (1003) of the slide section was analyzed with SlideBook image analysis software (SlideBook 5.0) on a Macintosh computer. Vessels in each section were defined by the circular or ovoid image of the brown endothelial walls. Capillaries, arterioles, and venules were counted. For determination of area, the minimum and maximum major axix of each vessel was measured. The calibration scale was set with stage micrometer to match computer pixels to a micrometer scale. Vessel count and vascular surface area estimates were reported per square millimeter of flap tissue area.
Statistical Analysis All data are expressed as the mean 6 SEM. Differences among groups and between baseline and subsequent time points were
determined with paired Student’s t-test and repeated-measures ANOVA with Tukey significant difference test used for post hoc analysis. A p value of 0.05 or less was considered statistically significant.
RESULTS Model of Graded Ischemia
Documented by FITC-dextran perfusion and fluorescent imaging, the blood supply to the skin and subcutaneous muscle of the mouse back is segmentally arranged and originated from intercostal and lumbar arteries that join in the midline (Fig. 2A). These vessels provide direct blood supply to the layers of the skin which in the mouse include the epidermis, dermis, subcutaneous fat, and panniculus carnosus muscle (Fig. 2B). Mouse back skin and subcutaneous tissue microvascular anatomy, elucidated by using CD-31 immunostaining to identify endothelium, is similar to human skin. The cutaneous vasculature is comprised of a superficial and deep vascular plexus connected by bridging capillaries. The deep vascular plexus is in continuity with perforating vessels within the panniculus carnosus muscle (Fig. 2C). Creation of the cranial-based, peninsular-shaped flap by two longitudinal incisions and connecting transverse incision produced a gradient of ischemia resulting from the interruption of segmental and underlying blood supply. FITC-dextran perfusion immediately following flap creation confirmed early ischemia of the distal flap (Fig. 2D).
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FIG. 2. (A) Representative whole mount image of the dorsal skin from a C57BL6 mouse following perfusion with FITC-dextran. Cranial and caudal directions as well as the dorsal midline (arrows) are indicated. (B) Histologic section of mouse back skin demonstrating composition of myocutaneous flap (H and E stain, bar in right lower corner ¼ 100 mm). (C) The microvascular anatomy of mouse back skin demonstrated using CD-31 immunostaining to identify endothelium (bar ¼ 100 mm). (D) FITC-dextran perfusion immediately following flap creation. Note the cranial to caudal gradient of perfusion.
MCGUIRE AND HOWDIESHELL: THE IMPORTANCE OF ENGRAFTMENT IN FLAP REVASCULARIZATION
Repositioning of the flap resulted in the development of a vascular-rich granulation tissue and the appearance of new vessels within the panniculus carnosus muscle layer (Fig. 3A). The positioning of a silicone sheet below the elevated flap resulted in less vascularized granulation tissue despite the appearance of active angiogenesis within the underlying deep fascia and paraspinal muscle layers (Fig. 3B). Gross evidence of distal flap necrosis was noted in all mice (n ¼ 6) with silicone, and four of these mice developed dehiscence of the distal flap from the adjacent back skin without evidence of seroma or infection. All of the flaps without silicone (n ¼ 6) healed without cutaneous necrosis or flap dehiscence. The complete incisional flap, created by a fourth transverse incision, was totally severed from surrounding tissues with an abrupt termination of its circulation. Despite return to the recipient bed, full thickness flap necrosis and eschar formation was evident at 2 wk following the establishment of the surgical wound. Brightfield and fluorescent microscopic examination of flap histologic sections confirmed and quantified the gradient of flap ischemia and the importance of engraftment in flap revascularization. Hematoxylin and eosin (H and E) staining of sections corresponding to each of the laser image ROIs (caudal, central, cranial) demonstrated a spectrum of viability ranging from minimal epidermolysis to full thickness myocutaneous necrosis, dependent on spatial location, time course, presence or absence of silicone, and complete incision. By day 7, epidermal regeneration with viable panniculus muscle
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was evident in the caudal region of flaps without silicone (Fig. 4A), while the identical region of flaps with silicone contained full thickness cutaneous necrosis and scattered muscle bundle necrosis illustrating the importance of engraftment in the revascularization of the flap (Fig. 4B). By day 3, extensive and irreversible myocutaneous necrosis was evident in the complete incisional flap, demonstrating the requirement of basal perfusion and engraftment for flap viability (Fig. 4C). Flap viability with and without engraftment was further analyzed using GFP transgenic mice. GFP expression in the skin and associated soft tissue was dominant in the panniculus carnosus muscle (Fig. 5A), making it a reproducible biomarker of flap viability, as ischemic and necrotic muscle fibers do not express GFP [10]. As early as day 3, there was a statistically significant decrease in GFP expression intensity in peninsular flaps of mice without silicone progressing in a cranial to caudal direction (Fig. 5B). The insertion of silicone significantly decreased the flap viability as indicated by the loss of GFP expression in the panniculus carnosus muscle layer due to lack of engraftment (Fig. 5B, C). At day 3, there was no demonstrable GFP expression above background in all (n ¼ 6) complete incisional flaps, consistent with necrosis due to absence of circulation (data not shown). Laser Speckle Perfusion Imaging
Prior to surgical intervention (control), mouse back skin perfusion was measured and recorded using the
FIG. 3. (A) Histologic section of flap without silicone. Note the granulation tissue neovessels bridging the paraspinal musculature with the flap panniculus carnosus muscle characteristic of engraftment (CD-31 immunostaining, bar ¼ 100 mm). (B) Histologic section of flap with silicone sheeting. Note lack of neovasculature bridging paraspinal musculature with panniculus muscle of flap due to silicone blockage of engraftment (CD-31 immunostaining, bar ¼ 100 mm) (arrow denotes position of silicone sheeting).
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FIG. 4. (A) Photomicrograph of the distal portion of a peninsular flap without silicone at day 7. Note viable epidermis, dermis, and panniculus carnosus muscle (H and E stain, bar ¼ 100 mm). (B) Photomicrograph of the distal portion of a peninsular flap with silicone at day 7 demonstrating epidermal and dermal necrosis and scattered panniculus muscle bundle necrosis (H and E stain, bar ¼ 100 mm). (C) Photomicrograph of the complete incisional flap at day 3 demonstrating full thickness myocutaneous necrosis (H and E stain, bar ¼ 100 mm).
FLPI instrument. Note consistent perfusion from cranial to caudal in each ROI (cranial, central, and caudal) region prior to flap creation (Fig. 6A, B). Immediately following flap creation, the flap was retracted in a cranial direction to allow measurement of perfusion of the underlying paraspinal muscle, revealing significantly higher deep muscle perfusion values compared with the overlying skin and panniculus muscle (Fig. 6C). To validate the depth of perfusion measurement, a sterile ultra-thin metallic ribbon retractor was placed between the flap and deep musculature to block any contribution to flap perfusion from the underlying paraspinal muscles. Perfusion measurements recorded with and without the retractor blockade were identical, confirming the ability of the instrument to discriminate the perfusion of the flap from underlying tissues. In addition, the silicone sheeting did not produce optical interference or an artifactual error in perfusion measurement as evidenced by comparable
immediate post-creation perfusion values for flaps with and without silicone at each ROI. Laser speckle imaging demonstrated significant spatial and temporal differences in perfusion between flaps with and without silicone. In both groups of mice, immediately following peninsular flap creation (day 0), flap perfusion decreased comparably in a cranial to caudal gradient (Fig. 7). In the group without silicone, progressive and sustained increases in flap perfusion were recorded in the cranial and central ROIs, with day 14 values greater than control values for each ROI. In the caudal ROI, flap perfusion increased over time, but did not reach control values (Fig. 7A, B). However, day 14 caudal flap perfusion was significantly greater than the perfusion measured immediately after flap creation (day 0). In contrast, in the silicone group of mice, flap perfusion recorded in the cranial and central ROIs fluctuated over time and did not reach control values at day 14
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FIG. 5. (A) Two channel fluorescent photomicrograph demonstrating constitutive GFP expression (green) in panniculus carnosus muscle in control back skin (DAPI nuclear stain: blue; bar ¼ 100 mm). (B) GFP fluorescence intensity versus flap spatial location. Note the significant gradient of GFP loss in both groups and the increased magnitude of the gradient in the silicone group due to lack of engraftment (*P < 0.05 versus cranial region of flap without silicone; **P < 0.05 versus identical region of flap without silicone; n ¼ 6 in each group). (C) Day 7 representative fluorescent photomicrographs of regions of a peninsular flap with silicone sheeting qualitatively demonstrating the cranial to caudal gradient of GFP loss in the panniculus carnosus muscle.
(Fig. 7C, D). In addition, caudal or distal flap perfusion did not vary significantly from immediate creation (day 0) values, suggesting impaired revascularization due to lack of engraftment. Laser speckle analysis of the complete incisional flap demonstrated immediate and uniform lack of perfusion across all ROIs, no return of perfusion over the 14 d interval, and marked hyperemia of the surrounding back skin, confirming the instrument’s sensitivity and specificity in determining flap perfusion (Fig. 8A, B). Microvascular Density Analysis
To correlate spatial and temporal flap perfusion with microvascular anatomy, CD-31 immunostaining and digital image analysis of control, day 3, 7, and 14, histologic sections was utilized to determine vessel count and vascular luminal cross-sectional surface area. By day 14, vessel counts in the caudal or distal portion of flaps without silicone sheeting were 3-fold greater than
control counts (Fig. 9A). Over the study period, vessel numbers in the cranial and central portions of these flaps remained essentially unchanged, while vascular surface area estimates increased 3- to 4-fold (Fig. 9B). Conversely, insertion of silicone sheeting, effectively blocking engraftment, resulted in a 2-fold reduction in distal flap vessel count, and corresponding reduction in vascular surface area. The vessel counts in the cranial and central regions of blocked flaps remained unchanged, with a limited increase in vascular surface area in the cranial flap region (Fig. 10A, B). Quantitation of vessel number and surface area correlated with morphology of the tissues as engraftment of flaps without silicone demonstrated revascularization via new vessel growth or neovascularization from deep musculature and dilatation of pre-existing proximal vessels (Fig. 3A; Fig. 11A, B). In contrast, prevention of engraftment with silicone resulted in limited dilatation of proximal flap vasculature and inhibition of distal neovascularization.
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FIG. 6. (A) Preoperative (control) laser speckle perfusion image of mouse back skin with caudal (A), central (B), and cranial (C) regions of interest (ROI) where quantitative measurements of perfusion were obtained. The color scale illustrates variations in perfusion from maximum (red) to minimum (blue). (B) Quantitative preoperative laser speckle perfusion (PU) versus flap spatial location. Note the consistent perfusion of back skin across regions, and the greater perfusion of underlying paraspinal musculature (*P < 0.05 versus back skin). (C) Laser speckle perfusion image immediately following flap creation with the flap elevated demonstrating the high perfusion of the paraspinal musculature. Color scale: maximal perfusion (red), minimal perfusion (blue).
DISCUSSION
We investigated a murine model of myocutaneous ischemia that was technically simple, created a distinct, two-dimensional gradient of ischemia, and allowed for identification of the origin of neovasculature. Laser speckle perfusion imaging accurately predicted the spatial and temporal pattern of ischemia, the return of functional revascularization, and the importance of engraftment in flap perfusion and survival. Histopathologic analysis and quantitative determination of microvascular density demonstrated engraftment resulted in flap revascularization via distal neovascularization from the recipient bed, and dilatation of the proximal pre-existing flap vasculature, while insertion of silicone sheeting, blocking engraftment, resulted in limited proximal vascular dilatation and inhibition of distal neovascularization.
Arteriogenesis is defined as the structural enlargement by growth of pre-existing arteriolar connections into true collateral arteries [11]. Collateral arteries can increase their lumen size by active proliferation and remodeling, thus increasing the capacity to carry blood to ischemic regions [12]. Angiogenesis is defined as the sprouting of pre-existing resident endothelial cells to form neovessels [13]. In contrast to angiogenesis, which is dominated by a single growth factor, vascular endothelial growth factor (VEGF), arteriogenesis relies on a complex interplay of many growth factors, cytokines, different cell types, a multitude of proteolytic enzymes, and at least during the initial stages, an environment of inflammation [14, 15]. Tissue ischemia/hypoxia is required for angiogenesis but not for arteriogenesis, which is governed initially by physical forces that activate the endothelium of pre-existing arterioles
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FIG. 7. (A) Quantitative laser speckle perfusion versus time of peninsular flaps without silicone demonstrating the gradient of ischemia between flap regions. At day 14, there is a significant increase in perfusion in all flap regions compared with immediate post-creation (day 0) values (*P < 0.05 versus day 0, n ¼ 6). C ¼ control or preoperative. (B) Laser speckle perfusion image of peninsular flap without silicone at day 7 demonstrating improved distal perfusion. The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue). (C) Quantitative laser speckle perfusion versus time of peninsular flaps with silicone. At day 14, there is no significant increase in perfusion compared with day 0 due to lack of engraftment (n ¼ 6). C ¼ control or pre-operative. (D) Laser speckle perfusion image of peninsular flap with silicone at day 7. There is improved distal perfusion compared with day 0, but reduced perfusion compared with the flap without silicone (compare with Fig. 9B). The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue).
[16]. After peripheral artery occlusion in rabbits and mice, arteriogenesis proceeds faster than angiogenesis because of a structural dilatation of pre-existing collateral vessels followed by mitosis of all vascular cell types, which restores resting blood flow within 3–5 d [17]. This study demonstrated that laser speckle perfusion imaging accurately discriminated the perfusion of a myocutaneous flap from the underlying tissue with high spatial resolution, and provided real-time and dynamic live imaging of the return of functional flap revascularization. Histologic analysis suggested that early proximal flap arteriogenesis and later distal flap angiogenesis are the processes responsible for revascularization of the engrafted flap. Over the last two decades, laser Doppler flowmetry (LDF) has been established as a routine experimental and clinical tool for noninvasive monitoring of blood flow velocity [18]. However, LDF is technically limited in that blood flow is only measured at a single point and at depths greater than skin thickness [19]. Only if
the LDF probe remains stationary during the measurement period can blood flow changes be measured in a comparative fashion, for example, before and after an intervention expected to cause variations in regional blood flow. The quality of these LDF measurements are termed ‘‘relative’’ since it is impossible to detect or quantify the absolute blood flow or to compare regional blood flow velocities over a large surface area [20]. As an advancement of conventional LDF, laser speckle contrast imaging was developed as a complementary ‘‘full-field’’ laser scanning technique to simultaneously obtain a map of blood flow velocity distribution over a larger and more superficial surface area and provide real-time images of perfusion [21]. Laser speckle is a random interference effect that occurs after an object is illuminated by laser light. Laser speckle contrast analysis exploits the fact that the random speckle pattern generated when tissue is illuminated by laser light changes when blood cells move within the region of interest. When there is a high level of movement (fast flow), the changing pattern becomes
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FIG. 8. (A) Laser speckle perfusion image of complete incisional flap immediately following flap creation (day 0) demonstrating lack of flap perfusion. The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue). (B) Laser speckle perfusion image of complete incisional flap at day 14. The limits of the surgical wound are indicated by arrowheads. Note lack of flap perfusion and marked increase in perfusion of surrounding back skin (red). Color scale: maximal perfusion (red), minimal perfusion (blue).
more blurred and the contrast in that region is reduced accordingly. Therefore, low contrast is related to high flow and high contrast to low flow [22]. The contrast image is processed to produce a color-coded live image that correlates with the perfusion in the tissue [23]. There are factors that affect the accuracy of laser speckle perfusion imaging including tissue temperature, motion, optical properties, curvature/angle, and scanning distance [18]. All measurements were performed at a constant ambient temperature and scanning distance. Images were captured in high speed mode and analyzed at end-expiration to limit motion artifact. Specular reflection, produced by varying surface curvature/angle, results in measurement artifact, as this component of the backscattered light has not entered the tissue and, therefore, contains no information about the underlying vasculature. The FLPI instrument takes advantage of the property that specular reflection from a polarized laser source is itself polarized, and utilizes a polarizer over the light collecting optics to eliminate this artifact [7].
FIG. 9. (A) Quantitative vessel count versus time of peninsular flap without silicone. Note the significant increase in number of vessels in the distal (caudal) region of the flap with time (*P < 0.05 versus control, n ¼ 6). C ¼ control or pre-operative. (B) Quantitative vascular surface area versus time of peninsular flaps without silicone demonstrating the progressive rise over time of luminal surface area in all flap regions (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative.
FIG. 10. (A) Quantitative vessel count versus time of peninsular flaps with silicone. Note the significant reduction in number of vessels in the distal (caudal) area in all flap regions (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative. (B) Quantitative vascular surface area versus time of peninsular flaps with silicone demonstrating a reduction over time of luminal surface area in the distal (caudal) flap region (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative.
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FIG. 11. (A) Representative histologic section of the cranial region of a peninsular flap without silicone at day 7. Note the markedly dilated flap subcutaneous blood vessel consistent with arteriogenesis (CD-31 immunostaining, bar ¼ 100 mm). (B) Histologic section of the cranial region of a peninsular flap without silicone at day 7. Note the markedly enlarged dermal vessels consistent with arteriogenesis (CD-31 immunostaining, bar ¼ 100 mm).
The establishment of functional revascularization is essential for the successful healing of cutaneous wounds, and is likely to involve the coordinated expression and activity of multiple factors, including angiogenic growth factors, chemokines, and stem cell recruitment factors [24–26]. Further studies utilizing this model and laser speckle technology will allow us to noninvasively monitor the spatial and temporal restitution of functional blood flow and potentially correlate this with biological markers of arteriogenesis and angiogenesis. Furthermore, a noninvasive method of mapping surface blood flow may make the cutaneous circulation a useful translational model for investigating mechanisms of revascularization and provide preclinical data about the state of microcirculatory function in high-risk populations, such as diabetes mellitus, obesity, arterial occlusive disease, and primary aging.
REFERENCES 1. Isner JM. Tissue responses to ischemia: Local and remote responses for preserving perfusion of ischemic muscle. J Clin Invest 2000;106:615. 2. Balasubramani M, Kumar TR, Babu M. Skin substitutes: A review. Burns 2001;27:534. 3. Schechner JS, Crane SK, Wang F. Engraftment of humanized skin equivalents. FASEB J 2003;17:2250. 4. Diaz-Sandoval LJ, Losordo DW. Gene therapy for cardiovascular angiogenesis. Expert Opin Biol Ther 2003;3:599. 5. Choksy SA, Chan P. Therapeutic angiogenesis. Br J Surg 2006; 93:261. 6. Barac A, Campia U, Panze JA. Methods for evaluating endothelial function in humans. Hypertension 2007;49:748.
7. Briers JD. Laser Doppler, speckle and related techniques for blood perfusion mapping and imaging. Physiol Meas 2001; 22:R35. 8. Choi B, Kang NM, Nelson JS. Laser speckle imaging for monitoring blood flow dynamics in the in vivo rodent dorsal skin fold model. Microvasc Res 2004;68:143. 9. Leahy MJ, Enfield JG, Clancy NT, et al. Biphotonic methods in microcirculation imaging. Medical Laser Application 2007;22:105. 10. Moloney TC, Dockery P, Windebank AJ, et al. Survival and immunogenicity of mesenchymal stem cells from the green fluorescent protein transgenic rodent. Neurorehab Neural Repair 2010; 12:1. 11. Heil M, Eitenmuller I, Schmitz-Rixen T, et al. Arteriogenesis versus angiogenesis: Similarities and differences. J Cell Mol Med 2006;10:45. 12. Unger EF, Sheffield CD, Epstein SE. Heparin promotes the formation of extracardiac to coronary anastomoses in a canine model. Am J Physiol 1991;260:H1625. 13. Scholz D, Ziegelhoeffer T, Helisch A, et al. Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice. J Mol Cell Cardiol 2002;34:775. 14. Schaper W, Scholz D. Factors regulating arteriogenesis. Arterioscler Thromb Vasc Biol 2003;23:1143. 15. Schaper W. Collateral circulation: Past and present. Basic Res Cardiol 2009;104:5. 16. Garcia-Cardena G, Comander J, Anderson KR, et al. Biomechanical activation of vascular endothelium as a determinant of its functional phenotype. Proc Natl Acad Sci USA 2001; 98:4478. 17. Shireman PK. The chemokine system in arteriogenesis and hindlimb ischemia. J Vasc Surg 2007;45:48A. 18. O’Doherty J, McNamara P, Clancy NT, et al. Comparison of instruments for investigation of microcirculatory blood flow and red blood cell concentration. J Biomed Optics 2009;14:1. 19. Oberg PA, Tenland T, Nilsson GE. Laser Doppler flowmetrya noninvasive and continuous method for blood flow evaluation in microvascular studies. Acta Med Scand Suppl 1984;687:17. 20. Arildsson ML, Wardell K, Nilsson GE. Higher order moment processing of laser Doppler perfusion signals. J Biomed Optics 1997;2:358.
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21. Fercher F, Briers JD. Flow visualization by means of singleexposure speckle photography. Opt Commun 1981;37:326. 22. O’Doherty J, Henricson J, Anderson C, et al. Subepidermal imaging using polarized light spectroscopy for assessment of skin microcirculation. Skin Res Technol 2007;13:472. 23. Nilsson GE, Zhai H, Farahmand S, et al. Cutaneous bioengineering instrumentation standardization: The tissue viability imager. Skin Res Technol 2009;15:6.
24. Carmeliet P. Angiogenesis in health and disease. Nature Med 2003;9:653. 25. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nature Rev Molec Cell Biol 2007; 8:464. 26. Van Royen N, Piek JJ, Schaper W, et al. A critical review of clinical arteriogenesis research. J Am Coll Cardiol 2010; 55:17.
The Importance of Engraftment in Flap Revascularization: Confirmation by Laser Speckle Perfusion Imaging Paul G. McGuire, Ph.D.,* and Thomas R. Howdieshell, M.D.†,1 *Department of Cell Biology and Physiology; and †Department of Surgery, University of New Mexico HSC, Albuquerque, New Mexico Submitted for publication June 20, 2010
Background. The delivery of proangiogenic agents in clinical trials of wound healing has produced equivocal results, the lack of real-time assessment of vascular growth is a major weakness in monitoring the efficacy of therapeutic angiogenesis, and surgical solutions fall short in addressing the deficiency in microvascular blood supply to ischemic wounds. Therefore, elucidation of the mechanisms involved in ischemiainduced blood vessel growth has potential diagnostic and therapeutic implications in wound healing. Materials and Methods. Three surgical models of wound ischemia, a cranial-based myocutaneous flap, an identical flap with underlying silicone sheeting to prevent engraftment, and a complete incisional flap without circulation were created on C57BL6 transgenic mice. Laser speckle contrast imaging was utilized to study the pattern of ischemia and return of revascularization. Simultaneous analysis of wound histology and microvascular density provided correlation of wound perfusion and morphology. Results. Creation of the peninsular-shaped flap produced a gradient of ischemia. Laser speckle contrast imaging accurately predicted the spatial and temporal pattern of ischemia, the return of functional revascularization, and the importance of engraftment in distal flap perfusion and survival. Histologic analysis demonstrated engraftment resulted in flap revascularization by new blood vessel growth from the recipient bed and dilatation of pre-existing flap vasculature. Conclusions. Further research utilizing this model of graded wound ischemia and the technology of laser speckle perfusion imaging will allow monitoring of the real-time restitution of blood flow for correlation with molecular biomarkers of revascularization in an
1 To whom correspondence and reprint requests should be addressed at Department of Surgery, MSC10-5610, University of New Mexico HSC, Albuquerque, NM 87131. E-mail: thowdieshell@salud. unm.edu.
attempt to gain further understanding of wound microvascular biology. Ó 2010 Elsevier Inc. All rights reserved. Key Words: wound healing; ischemia; laser speckle perfusion imaging; revascularization; arteriogenesis; angiogenesis. INTRODUCTION
One of the most potent stimuli for new blood vessel growth or neovascularization is ischemia, which induces capillary growth to restore adequate oxygen delivery to hypoxic tissues. Ischemia initiates a number of angiogenic processes, including the release of cytokines and growth factors, the up-regulation of extracellular proteinases and cell surface molecules, and the proliferation of mature endothelial cells [1]. Avascular tissue-engineered skin equivalents have been available for years, and are used to treat wounds due to burns, trauma, surgical excision, nonhealing diabetic ulcers, and blistering skin diseases [2]. Although these products may improve wound healing, long term engraftment has not been demonstrated. It is likely that inadequate perfusion in the post-transplantation period is responsible for lack of engraftment. Whereas autologous split-thickness skin grafts with an inherent vasculature purportedly become perfused in a matter of days by direct anastomosis or inosculation of preexisting graft vessels with those of the recipient, avascular skin equivalents must become perfused entirely by neovascularization from the recipient wound bed [3]. Therefore, knowledge of the basic mechanisms involved in cutaneous revascularization is imperative for development of potential treatments which promote successful wound healing. There is avid interest in developing techniques to promote neovascularization in ischemic diseases such as peripheral arterial occlusive disease, coronary artery
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disease, and cerebrovascular disease [4]. Unfortunately, the delivery of proangiogenic agents in ongoing clinical trials has produced equivocal results, the lack of real-time assessment of vascular growth is a major weakness in monitoring the efficacy of therapeutic angiogenesis, and surgical solutions fall short in addressing the deficiency in microvascular blood supply of ischemic wounds [5, 6]. The recent commercial release (2007) of a new device, the Full-Field Laser Perfusion Imager (FLPI) from Moor Instruments, Essex, UK, offers a noninvasive and noncontact method of mapping flow fields such as capillary blood flow based on the principle of laser scatter contrast imaging. The technique works by illuminating an area of tissue with laser light to produce a high contrast random interference effect know as a speckle pattern [7]. Compared with laser Doppler imaging, the effective sampling depth of laser speckle imaging is less than 1 mm, and the resulting image is of blood flow in the microvessels of the surface layers of the tissue being sampled, for example the skin. The Moor FLPI system provides video frame rate images of microvascular flow, up to 25 images per second. Therefore, it is ideally suited to any application in which dynamic changes are occurring, such as during the cardiac or respiratory cycle, and conventional laser Doppler imaging cannot provide data with sufficient time resolution [8]. In the present study, we evaluated laser speckle contrast imaging as a method to predict the spatial and temporal pattern of wound ischemia and return of functional revascularization with corroboration by simultaneous histopathologic analysis and quantitative determination of microvascular density by CD-31 immunostaining. MATERIALS AND METHODS Mouse Myocutaneous Flap Model All animals were treated humanely in accordance with the National Research Council’s ‘‘Guide for the care and use of laboratory animals’’ as part of a protocol approved by the University of New Mexico’s animal review committee. Three surgical models of wound healing were created: a cranial-based myocutaneous flap, an identical flap with underlying silicone sheeting, and a complete incisional flap (Fig. 1). Female C57BL6 transgenic green fluorescent protein (GFP) mice, ages 4 to 6 mo, underwent anesthesia with isoflurane (1%–3%) via nose cone inhalation. Post-surgical analgesia was provided using a single subcutaneous dose (0.01 mg/kg) of buprenorphine hydrochloride. After maintenance of inhalation anesthesia, hair was removed from the mouse back skin using electric clippers, and the site was prepped with povidone-iodine and 70% ethanol. The first group of mice (n ¼ 6) underwent surgical creation of a peninsular flap (3 cm in length and 1.5 cm in width) consisting of skin, adipose tissue, and panniculus carnosus muscle by making three soft tissue incisions. The flap was elevated cranially and re-approximated to the back skin with 6-0 monofilament sutures. The silicone group of mice (n ¼ 6) underwent insertion of a 0.005 inch-thick silicone sheet (BioPlexus Corporation, Saticoy, CA) beneath the flap to separate the flap from the underlying
tissue bed to prevent engraftment. Finally, in a third group of mice (n ¼ 6), a complete incisional flap was created by making a fourth and transverse soft tissue incision resulting in complete separation of the flap from the underlying tissue prior to suture reapproximation. After surgery, each animal was singly housed and received water and food ad libitum.
Fluorescein Isothiocyanate (FITC)-Dextran Perfusion Mice were anesthetized with isoflurane and were perfused via the left ventricle with 30 mL of warm phosphate buffered saline (PBS) followed by 30 mL of 10% formalin containing 20 mg/mL FITC-dextran (Sigma Chemical, St. Louis, MO). Following perfusion, the back skin containing the flap was excised, placed dermal-side up, and examined using a fluorescence dissecting microscope (Leica MZ FLIII, Wetzlar, Germany).
Laser Speckle Perfusion Imaging On days 0, 1, 3, 5, 7, 10, and 14 after surgery, each animal underwent inhalation anesthesia in prone position, and laser speckle perfusion imaging was performed with the FLPI (Full-Field Laser Perfusion Imager; Moor Instruments, Essex, UK) in low resolution/ high speed setting at a display rate of 25 Hz, time constant of 1.0 s, and camera exposure time of 20 ms. The instrument head containing the CCD (charged coupled device) camera was positioned 30 cm above the mouse back tissue surface using an articulating arm. Real-time data was acquired in the live image measurement mode. The contrast images were processed to produce a scaled color-coded live flux image (red equaled high perfusion, blue equaled low perfusion), which correlated with the blood flow velocity in the tissue. The FLPI instrument reports perfusion in arbitrary units (PU). To assign values to a measurement, the imager was calibrated using a reference flux signal which was generated by the laser light scattered from a suspension of polystyrene microspheres in water undergoing thermal or Brownian motion. From kinetic theory, the average velocity of the microspheres is proportional to the square root of the temperature in Kelvin. All measurements were performed at a room temperature of 20 C (293 Kelvin) [9]. For each time point examined, 10 single frame images acquired at end expiration (no torso movement) were analyzed in the repeat image measurement window utilizing three identical regions of interest (ROI): caudal, central, and cranial. At each time point, the mean perfusion in each ROI was calculated for control skin and deep muscle, the peninsular flap with and without silicone sheeting, and the complete incisional flap.
Tissue Harvest, Histology, GFP Intensity Expression, and Immunochemical Staining Mice were sacrificed by CO2 inhalation on days 0 (control), 3, 7, and 14 for flap harvest and histologic examination. The entire flap was excised and sectioned in transverse fashion to directly correspond to the caudal, central, and cranial laser image ROIs. The tissue was fixed in IHC Zinc Fixative (BD Biosciences-Pharmingen, San Diego, CA) for 24 h, processed, and paraffin embedded. Serial sections (4 mm) were dewaxed in xylene, taken through graded ethanol, and then hydrated in PBS solution for fluorescent and brightfield microscopy. Caudal, central, and cranial sections were mounted using ProLong Gold Antifade Reagent with DAPI (Invitrogen-Molecular Probes, Eugene, OR) for fluorescent examination. DAPI (4’,6-diamino-2phenylindole) nuclear staining provided definition for tissue orientation. Sections were examined and photographed using two-channel fluorescent microscopy at 1003 magnification (Axioskop; Carl Zeiss Microimaging, Thornwood, NY). Captured images (475 nm excitation, 509 nm emission) were analyzed for GFP intensity expression using SlideBook digital microscopy software (SlideBook 5.0, Intelligent Imaging Innovations, Santa Monica, CA). A two-dimension image mask was created, the panniculus carnosus muscle was isolated and
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FIG. 1. Proposed surgical models. (A) The peninsular-shaped, myocutaneous flap. (B) The identical flap with underlying silicone sheeting to prevent interaction of the flap with the deep fascia and back musculature. (C) The complete incisional flap without circulation. highlighted using the paint brush feature, and the software was utilized to calculate the fluorescent emission intensity. Blood vessel identity was determined by CD-31 immunostaining. Following dewaxing and hydration, sections were incubated for 10 min in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, washed in PBS, and incubated with primary antibody (rat anti-mouse CD-31, 1:50, BD Biosciences-Pharmingen) for 1 h at room temperature in a humidified chamber. Next, the sections were incubated with a biotinylated secondary antibody (anti-rat Ig HRP kit, 1:50, BD Bioscience-Pharmingen) for 30 min at room temperature. The streptavidin-HRP reagent was applied for 30 min followed by the DAB chromogen for 5 min. The sections were counterstained with Vector Hematoxylin QS (Vector Laboratories, Burlingame, CA) with quick immersion. The 3,3’-diaminobenzidine (DAB) substrate-chromogen resulted in a brown-colored precipitate at the antigen site.
Microvascular Density Determination Skin, subcutaneous tissue, and panniculus muscle vessel count and vascular luminal cross-sectional surface areas were determined with image analysis of CD-31 immunostained sections. Multiple, consistent full thickness flap biopsies were analyzed for each caudal, central, and cranial section with mean values reported. A Zeiss microscope with attached digital camera was used for image acquisition. The magnified image (1003) of the slide section was analyzed with SlideBook image analysis software (SlideBook 5.0) on a Macintosh computer. Vessels in each section were defined by the circular or ovoid image of the brown endothelial walls. Capillaries, arterioles, and venules were counted. For determination of area, the minimum and maximum major axix of each vessel was measured. The calibration scale was set with stage micrometer to match computer pixels to a micrometer scale. Vessel count and vascular surface area estimates were reported per square millimeter of flap tissue area.
Statistical Analysis All data are expressed as the mean 6 SEM. Differences among groups and between baseline and subsequent time points were
determined with paired Student’s t-test and repeated-measures ANOVA with Tukey significant difference test used for post hoc analysis. A p value of 0.05 or less was considered statistically significant.
RESULTS Model of Graded Ischemia
Documented by FITC-dextran perfusion and fluorescent imaging, the blood supply to the skin and subcutaneous muscle of the mouse back is segmentally arranged and originated from intercostal and lumbar arteries that join in the midline (Fig. 2A). These vessels provide direct blood supply to the layers of the skin which in the mouse include the epidermis, dermis, subcutaneous fat, and panniculus carnosus muscle (Fig. 2B). Mouse back skin and subcutaneous tissue microvascular anatomy, elucidated by using CD-31 immunostaining to identify endothelium, is similar to human skin. The cutaneous vasculature is comprised of a superficial and deep vascular plexus connected by bridging capillaries. The deep vascular plexus is in continuity with perforating vessels within the panniculus carnosus muscle (Fig. 2C). Creation of the cranial-based, peninsular-shaped flap by two longitudinal incisions and connecting transverse incision produced a gradient of ischemia resulting from the interruption of segmental and underlying blood supply. FITC-dextran perfusion immediately following flap creation confirmed early ischemia of the distal flap (Fig. 2D).
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FIG. 2. (A) Representative whole mount image of the dorsal skin from a C57BL6 mouse following perfusion with FITC-dextran. Cranial and caudal directions as well as the dorsal midline (arrows) are indicated. (B) Histologic section of mouse back skin demonstrating composition of myocutaneous flap (H and E stain, bar in right lower corner ¼ 100 mm). (C) The microvascular anatomy of mouse back skin demonstrated using CD-31 immunostaining to identify endothelium (bar ¼ 100 mm). (D) FITC-dextran perfusion immediately following flap creation. Note the cranial to caudal gradient of perfusion.
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Repositioning of the flap resulted in the development of a vascular-rich granulation tissue and the appearance of new vessels within the panniculus carnosus muscle layer (Fig. 3A). The positioning of a silicone sheet below the elevated flap resulted in less vascularized granulation tissue despite the appearance of active angiogenesis within the underlying deep fascia and paraspinal muscle layers (Fig. 3B). Gross evidence of distal flap necrosis was noted in all mice (n ¼ 6) with silicone, and four of these mice developed dehiscence of the distal flap from the adjacent back skin without evidence of seroma or infection. All of the flaps without silicone (n ¼ 6) healed without cutaneous necrosis or flap dehiscence. The complete incisional flap, created by a fourth transverse incision, was totally severed from surrounding tissues with an abrupt termination of its circulation. Despite return to the recipient bed, full thickness flap necrosis and eschar formation was evident at 2 wk following the establishment of the surgical wound. Brightfield and fluorescent microscopic examination of flap histologic sections confirmed and quantified the gradient of flap ischemia and the importance of engraftment in flap revascularization. Hematoxylin and eosin (H and E) staining of sections corresponding to each of the laser image ROIs (caudal, central, cranial) demonstrated a spectrum of viability ranging from minimal epidermolysis to full thickness myocutaneous necrosis, dependent on spatial location, time course, presence or absence of silicone, and complete incision. By day 7, epidermal regeneration with viable panniculus muscle
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was evident in the caudal region of flaps without silicone (Fig. 4A), while the identical region of flaps with silicone contained full thickness cutaneous necrosis and scattered muscle bundle necrosis illustrating the importance of engraftment in the revascularization of the flap (Fig. 4B). By day 3, extensive and irreversible myocutaneous necrosis was evident in the complete incisional flap, demonstrating the requirement of basal perfusion and engraftment for flap viability (Fig. 4C). Flap viability with and without engraftment was further analyzed using GFP transgenic mice. GFP expression in the skin and associated soft tissue was dominant in the panniculus carnosus muscle (Fig. 5A), making it a reproducible biomarker of flap viability, as ischemic and necrotic muscle fibers do not express GFP [10]. As early as day 3, there was a statistically significant decrease in GFP expression intensity in peninsular flaps of mice without silicone progressing in a cranial to caudal direction (Fig. 5B). The insertion of silicone significantly decreased the flap viability as indicated by the loss of GFP expression in the panniculus carnosus muscle layer due to lack of engraftment (Fig. 5B, C). At day 3, there was no demonstrable GFP expression above background in all (n ¼ 6) complete incisional flaps, consistent with necrosis due to absence of circulation (data not shown). Laser Speckle Perfusion Imaging
Prior to surgical intervention (control), mouse back skin perfusion was measured and recorded using the
FIG. 3. (A) Histologic section of flap without silicone. Note the granulation tissue neovessels bridging the paraspinal musculature with the flap panniculus carnosus muscle characteristic of engraftment (CD-31 immunostaining, bar ¼ 100 mm). (B) Histologic section of flap with silicone sheeting. Note lack of neovasculature bridging paraspinal musculature with panniculus muscle of flap due to silicone blockage of engraftment (CD-31 immunostaining, bar ¼ 100 mm) (arrow denotes position of silicone sheeting).
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FIG. 4. (A) Photomicrograph of the distal portion of a peninsular flap without silicone at day 7. Note viable epidermis, dermis, and panniculus carnosus muscle (H and E stain, bar ¼ 100 mm). (B) Photomicrograph of the distal portion of a peninsular flap with silicone at day 7 demonstrating epidermal and dermal necrosis and scattered panniculus muscle bundle necrosis (H and E stain, bar ¼ 100 mm). (C) Photomicrograph of the complete incisional flap at day 3 demonstrating full thickness myocutaneous necrosis (H and E stain, bar ¼ 100 mm).
FLPI instrument. Note consistent perfusion from cranial to caudal in each ROI (cranial, central, and caudal) region prior to flap creation (Fig. 6A, B). Immediately following flap creation, the flap was retracted in a cranial direction to allow measurement of perfusion of the underlying paraspinal muscle, revealing significantly higher deep muscle perfusion values compared with the overlying skin and panniculus muscle (Fig. 6C). To validate the depth of perfusion measurement, a sterile ultra-thin metallic ribbon retractor was placed between the flap and deep musculature to block any contribution to flap perfusion from the underlying paraspinal muscles. Perfusion measurements recorded with and without the retractor blockade were identical, confirming the ability of the instrument to discriminate the perfusion of the flap from underlying tissues. In addition, the silicone sheeting did not produce optical interference or an artifactual error in perfusion measurement as evidenced by comparable
immediate post-creation perfusion values for flaps with and without silicone at each ROI. Laser speckle imaging demonstrated significant spatial and temporal differences in perfusion between flaps with and without silicone. In both groups of mice, immediately following peninsular flap creation (day 0), flap perfusion decreased comparably in a cranial to caudal gradient (Fig. 7). In the group without silicone, progressive and sustained increases in flap perfusion were recorded in the cranial and central ROIs, with day 14 values greater than control values for each ROI. In the caudal ROI, flap perfusion increased over time, but did not reach control values (Fig. 7A, B). However, day 14 caudal flap perfusion was significantly greater than the perfusion measured immediately after flap creation (day 0). In contrast, in the silicone group of mice, flap perfusion recorded in the cranial and central ROIs fluctuated over time and did not reach control values at day 14
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FIG. 5. (A) Two channel fluorescent photomicrograph demonstrating constitutive GFP expression (green) in panniculus carnosus muscle in control back skin (DAPI nuclear stain: blue; bar ¼ 100 mm). (B) GFP fluorescence intensity versus flap spatial location. Note the significant gradient of GFP loss in both groups and the increased magnitude of the gradient in the silicone group due to lack of engraftment (*P < 0.05 versus cranial region of flap without silicone; **P < 0.05 versus identical region of flap without silicone; n ¼ 6 in each group). (C) Day 7 representative fluorescent photomicrographs of regions of a peninsular flap with silicone sheeting qualitatively demonstrating the cranial to caudal gradient of GFP loss in the panniculus carnosus muscle.
(Fig. 7C, D). In addition, caudal or distal flap perfusion did not vary significantly from immediate creation (day 0) values, suggesting impaired revascularization due to lack of engraftment. Laser speckle analysis of the complete incisional flap demonstrated immediate and uniform lack of perfusion across all ROIs, no return of perfusion over the 14 d interval, and marked hyperemia of the surrounding back skin, confirming the instrument’s sensitivity and specificity in determining flap perfusion (Fig. 8A, B). Microvascular Density Analysis
To correlate spatial and temporal flap perfusion with microvascular anatomy, CD-31 immunostaining and digital image analysis of control, day 3, 7, and 14, histologic sections was utilized to determine vessel count and vascular luminal cross-sectional surface area. By day 14, vessel counts in the caudal or distal portion of flaps without silicone sheeting were 3-fold greater than
control counts (Fig. 9A). Over the study period, vessel numbers in the cranial and central portions of these flaps remained essentially unchanged, while vascular surface area estimates increased 3- to 4-fold (Fig. 9B). Conversely, insertion of silicone sheeting, effectively blocking engraftment, resulted in a 2-fold reduction in distal flap vessel count, and corresponding reduction in vascular surface area. The vessel counts in the cranial and central regions of blocked flaps remained unchanged, with a limited increase in vascular surface area in the cranial flap region (Fig. 10A, B). Quantitation of vessel number and surface area correlated with morphology of the tissues as engraftment of flaps without silicone demonstrated revascularization via new vessel growth or neovascularization from deep musculature and dilatation of pre-existing proximal vessels (Fig. 3A; Fig. 11A, B). In contrast, prevention of engraftment with silicone resulted in limited dilatation of proximal flap vasculature and inhibition of distal neovascularization.
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FIG. 6. (A) Preoperative (control) laser speckle perfusion image of mouse back skin with caudal (A), central (B), and cranial (C) regions of interest (ROI) where quantitative measurements of perfusion were obtained. The color scale illustrates variations in perfusion from maximum (red) to minimum (blue). (B) Quantitative preoperative laser speckle perfusion (PU) versus flap spatial location. Note the consistent perfusion of back skin across regions, and the greater perfusion of underlying paraspinal musculature (*P < 0.05 versus back skin). (C) Laser speckle perfusion image immediately following flap creation with the flap elevated demonstrating the high perfusion of the paraspinal musculature. Color scale: maximal perfusion (red), minimal perfusion (blue).
DISCUSSION
We investigated a murine model of myocutaneous ischemia that was technically simple, created a distinct, two-dimensional gradient of ischemia, and allowed for identification of the origin of neovasculature. Laser speckle perfusion imaging accurately predicted the spatial and temporal pattern of ischemia, the return of functional revascularization, and the importance of engraftment in flap perfusion and survival. Histopathologic analysis and quantitative determination of microvascular density demonstrated engraftment resulted in flap revascularization via distal neovascularization from the recipient bed, and dilatation of the proximal pre-existing flap vasculature, while insertion of silicone sheeting, blocking engraftment, resulted in limited proximal vascular dilatation and inhibition of distal neovascularization.
Arteriogenesis is defined as the structural enlargement by growth of pre-existing arteriolar connections into true collateral arteries [11]. Collateral arteries can increase their lumen size by active proliferation and remodeling, thus increasing the capacity to carry blood to ischemic regions [12]. Angiogenesis is defined as the sprouting of pre-existing resident endothelial cells to form neovessels [13]. In contrast to angiogenesis, which is dominated by a single growth factor, vascular endothelial growth factor (VEGF), arteriogenesis relies on a complex interplay of many growth factors, cytokines, different cell types, a multitude of proteolytic enzymes, and at least during the initial stages, an environment of inflammation [14, 15]. Tissue ischemia/hypoxia is required for angiogenesis but not for arteriogenesis, which is governed initially by physical forces that activate the endothelium of pre-existing arterioles
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FIG. 7. (A) Quantitative laser speckle perfusion versus time of peninsular flaps without silicone demonstrating the gradient of ischemia between flap regions. At day 14, there is a significant increase in perfusion in all flap regions compared with immediate post-creation (day 0) values (*P < 0.05 versus day 0, n ¼ 6). C ¼ control or preoperative. (B) Laser speckle perfusion image of peninsular flap without silicone at day 7 demonstrating improved distal perfusion. The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue). (C) Quantitative laser speckle perfusion versus time of peninsular flaps with silicone. At day 14, there is no significant increase in perfusion compared with day 0 due to lack of engraftment (n ¼ 6). C ¼ control or pre-operative. (D) Laser speckle perfusion image of peninsular flap with silicone at day 7. There is improved distal perfusion compared with day 0, but reduced perfusion compared with the flap without silicone (compare with Fig. 9B). The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue).
[16]. After peripheral artery occlusion in rabbits and mice, arteriogenesis proceeds faster than angiogenesis because of a structural dilatation of pre-existing collateral vessels followed by mitosis of all vascular cell types, which restores resting blood flow within 3–5 d [17]. This study demonstrated that laser speckle perfusion imaging accurately discriminated the perfusion of a myocutaneous flap from the underlying tissue with high spatial resolution, and provided real-time and dynamic live imaging of the return of functional flap revascularization. Histologic analysis suggested that early proximal flap arteriogenesis and later distal flap angiogenesis are the processes responsible for revascularization of the engrafted flap. Over the last two decades, laser Doppler flowmetry (LDF) has been established as a routine experimental and clinical tool for noninvasive monitoring of blood flow velocity [18]. However, LDF is technically limited in that blood flow is only measured at a single point and at depths greater than skin thickness [19]. Only if
the LDF probe remains stationary during the measurement period can blood flow changes be measured in a comparative fashion, for example, before and after an intervention expected to cause variations in regional blood flow. The quality of these LDF measurements are termed ‘‘relative’’ since it is impossible to detect or quantify the absolute blood flow or to compare regional blood flow velocities over a large surface area [20]. As an advancement of conventional LDF, laser speckle contrast imaging was developed as a complementary ‘‘full-field’’ laser scanning technique to simultaneously obtain a map of blood flow velocity distribution over a larger and more superficial surface area and provide real-time images of perfusion [21]. Laser speckle is a random interference effect that occurs after an object is illuminated by laser light. Laser speckle contrast analysis exploits the fact that the random speckle pattern generated when tissue is illuminated by laser light changes when blood cells move within the region of interest. When there is a high level of movement (fast flow), the changing pattern becomes
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FIG. 8. (A) Laser speckle perfusion image of complete incisional flap immediately following flap creation (day 0) demonstrating lack of flap perfusion. The limits of the surgical wound are indicated by arrowheads. Color scale: maximal perfusion (red), minimal perfusion (blue). (B) Laser speckle perfusion image of complete incisional flap at day 14. The limits of the surgical wound are indicated by arrowheads. Note lack of flap perfusion and marked increase in perfusion of surrounding back skin (red). Color scale: maximal perfusion (red), minimal perfusion (blue).
more blurred and the contrast in that region is reduced accordingly. Therefore, low contrast is related to high flow and high contrast to low flow [22]. The contrast image is processed to produce a color-coded live image that correlates with the perfusion in the tissue [23]. There are factors that affect the accuracy of laser speckle perfusion imaging including tissue temperature, motion, optical properties, curvature/angle, and scanning distance [18]. All measurements were performed at a constant ambient temperature and scanning distance. Images were captured in high speed mode and analyzed at end-expiration to limit motion artifact. Specular reflection, produced by varying surface curvature/angle, results in measurement artifact, as this component of the backscattered light has not entered the tissue and, therefore, contains no information about the underlying vasculature. The FLPI instrument takes advantage of the property that specular reflection from a polarized laser source is itself polarized, and utilizes a polarizer over the light collecting optics to eliminate this artifact [7].
FIG. 9. (A) Quantitative vessel count versus time of peninsular flap without silicone. Note the significant increase in number of vessels in the distal (caudal) region of the flap with time (*P < 0.05 versus control, n ¼ 6). C ¼ control or pre-operative. (B) Quantitative vascular surface area versus time of peninsular flaps without silicone demonstrating the progressive rise over time of luminal surface area in all flap regions (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative.
FIG. 10. (A) Quantitative vessel count versus time of peninsular flaps with silicone. Note the significant reduction in number of vessels in the distal (caudal) area in all flap regions (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative. (B) Quantitative vascular surface area versus time of peninsular flaps with silicone demonstrating a reduction over time of luminal surface area in the distal (caudal) flap region (*P < 0.05 versus control, n ¼ 6). C ¼ control or preoperative.
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FIG. 11. (A) Representative histologic section of the cranial region of a peninsular flap without silicone at day 7. Note the markedly dilated flap subcutaneous blood vessel consistent with arteriogenesis (CD-31 immunostaining, bar ¼ 100 mm). (B) Histologic section of the cranial region of a peninsular flap without silicone at day 7. Note the markedly enlarged dermal vessels consistent with arteriogenesis (CD-31 immunostaining, bar ¼ 100 mm).
The establishment of functional revascularization is essential for the successful healing of cutaneous wounds, and is likely to involve the coordinated expression and activity of multiple factors, including angiogenic growth factors, chemokines, and stem cell recruitment factors [24–26]. Further studies utilizing this model and laser speckle technology will allow us to noninvasively monitor the spatial and temporal restitution of functional blood flow and potentially correlate this with biological markers of arteriogenesis and angiogenesis. Furthermore, a noninvasive method of mapping surface blood flow may make the cutaneous circulation a useful translational model for investigating mechanisms of revascularization and provide preclinical data about the state of microcirculatory function in high-risk populations, such as diabetes mellitus, obesity, arterial occlusive disease, and primary aging.
REFERENCES 1. Isner JM. Tissue responses to ischemia: Local and remote responses for preserving perfusion of ischemic muscle. J Clin Invest 2000;106:615. 2. Balasubramani M, Kumar TR, Babu M. Skin substitutes: A review. Burns 2001;27:534. 3. Schechner JS, Crane SK, Wang F. Engraftment of humanized skin equivalents. FASEB J 2003;17:2250. 4. Diaz-Sandoval LJ, Losordo DW. Gene therapy for cardiovascular angiogenesis. Expert Opin Biol Ther 2003;3:599. 5. Choksy SA, Chan P. Therapeutic angiogenesis. Br J Surg 2006; 93:261. 6. Barac A, Campia U, Panze JA. Methods for evaluating endothelial function in humans. Hypertension 2007;49:748.
7. Briers JD. Laser Doppler, speckle and related techniques for blood perfusion mapping and imaging. Physiol Meas 2001; 22:R35. 8. Choi B, Kang NM, Nelson JS. Laser speckle imaging for monitoring blood flow dynamics in the in vivo rodent dorsal skin fold model. Microvasc Res 2004;68:143. 9. Leahy MJ, Enfield JG, Clancy NT, et al. Biphotonic methods in microcirculation imaging. Medical Laser Application 2007;22:105. 10. Moloney TC, Dockery P, Windebank AJ, et al. Survival and immunogenicity of mesenchymal stem cells from the green fluorescent protein transgenic rodent. Neurorehab Neural Repair 2010; 12:1. 11. Heil M, Eitenmuller I, Schmitz-Rixen T, et al. Arteriogenesis versus angiogenesis: Similarities and differences. J Cell Mol Med 2006;10:45. 12. Unger EF, Sheffield CD, Epstein SE. Heparin promotes the formation of extracardiac to coronary anastomoses in a canine model. Am J Physiol 1991;260:H1625. 13. Scholz D, Ziegelhoeffer T, Helisch A, et al. Contribution of arteriogenesis and angiogenesis to postocclusive hindlimb perfusion in mice. J Mol Cell Cardiol 2002;34:775. 14. Schaper W, Scholz D. Factors regulating arteriogenesis. Arterioscler Thromb Vasc Biol 2003;23:1143. 15. Schaper W. Collateral circulation: Past and present. Basic Res Cardiol 2009;104:5. 16. Garcia-Cardena G, Comander J, Anderson KR, et al. Biomechanical activation of vascular endothelium as a determinant of its functional phenotype. Proc Natl Acad Sci USA 2001; 98:4478. 17. Shireman PK. The chemokine system in arteriogenesis and hindlimb ischemia. J Vasc Surg 2007;45:48A. 18. O’Doherty J, McNamara P, Clancy NT, et al. Comparison of instruments for investigation of microcirculatory blood flow and red blood cell concentration. J Biomed Optics 2009;14:1. 19. Oberg PA, Tenland T, Nilsson GE. Laser Doppler flowmetrya noninvasive and continuous method for blood flow evaluation in microvascular studies. Acta Med Scand Suppl 1984;687:17. 20. Arildsson ML, Wardell K, Nilsson GE. Higher order moment processing of laser Doppler perfusion signals. J Biomed Optics 1997;2:358.
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21. Fercher F, Briers JD. Flow visualization by means of singleexposure speckle photography. Opt Commun 1981;37:326. 22. O’Doherty J, Henricson J, Anderson C, et al. Subepidermal imaging using polarized light spectroscopy for assessment of skin microcirculation. Skin Res Technol 2007;13:472. 23. Nilsson GE, Zhai H, Farahmand S, et al. Cutaneous bioengineering instrumentation standardization: The tissue viability imager. Skin Res Technol 2009;15:6.
24. Carmeliet P. Angiogenesis in health and disease. Nature Med 2003;9:653. 25. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nature Rev Molec Cell Biol 2007; 8:464. 26. Van Royen N, Piek JJ, Schaper W, et al. A critical review of clinical arteriogenesis research. J Am Coll Cardiol 2010; 55:17.