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In vivo reflectance-mode confocal microscopy provides insights in human skin microcirculation and histomorphology
Computerized Medical Imaging and Graphics 33 (2009) 532–536
Contents lists available at ScienceDirect
Computerized Medical Imaging and Graphics journal homepage: www.elsevier.com/locate/compmedimag
In vivo reflectance-mode confocal microscopy provides insights in human skin microcirculation and histomorphology M.A. Altintas a,∗ , M. Meyer-Marcotty a , A.A. Altintas b , M. Guggenheim c , A. Gohritz a , M.C. Aust a , P.M. Vogt a a b c
Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany Department of Plastic and Reconstructive Surgery, Cologne-Merheim University of Witten-Herdecke, Cologne, Germany Division of Plastic and Reconstructive Surgery, Burn Centre, Department of Surgery, University Hospital Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 11 January 2009 Received in revised form 17 April 2009 Accepted 20 April 2009 Keywords: Non-invasive imaging High resolution imaging Vasoconstriction Vasodilatation Blood flow
a b s t r a c t Purpose: Various approaches are used to study microcirculation, however, no modality evaluates microcirculation and histomorphology on cellular levels. We hypothesized that reflectance-mode confocal microscopy (RCM) enables simultaneous evaluation in vivo of both microcirculation and histomorphology. Principals: The forearm of 20 volunteers was exposed to either local heat stress (HS-group), or to local cold stress (CS-group). RCM was performed prior and after temperature stress to evaluate quantitative blood-cell flow, capillary loop diameter, granular cell size, and basal layer thickness. Results: In the HS-group, we observed significant increase in capillary loop diameter and increased bloodcell flow after heat stress. In the CS-group, significant decreases of capillary loop diameter and in bloodcell flow were determined following cold stress. Granular cell size and basal layer thickness differed insignificantly prior and after local temperature stress. Conclusions: RCM provides real-time and in vivo high resolution imaging of temperature-dependent changes in the human skin microcirculation and histomorphology on cellular levels. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction In the past years, various technical developments were evaluated to observe the human skin microcirculation in vivo, such as Laser Doppler Flowmetry [1], hydrogen clearance [2], near infrared spectroscopy [3], hemoglobin oxygenation [4] and pulsoxymetry [5]. However, none of these methods can sufficiently evaluate the dynamic microcirculation and simultaneously elucidate the cellular morphology and integrity. However, this information should be considered as irreplaceable to study the physiology and physiopathology of the skin efficiently. Thus, a high resolution imaging technique to visualize the human skin microcirculation and the associated cellular morphology would be novel in microcirculation research. Reflectance-mode confocal microscopy (RCM) enables real-time and in vivo high resolution imaging of the human skin and has been used in numerous studies in dermatology and the
burn care arena [6–10]. For correlation, confocal images were compared to histological sectioning and a high sensitivity of 82.9% and specificity of 95.7% was found [9,10]. The dynamic effects of temperature changes on the human skin microcirculation are well investigated using non-invasive approaches and also well described in the actual literature [11–16]. Temperature-dependent microcirculation changes have been studied, but only at temperatures that damage the skin, using more complex techniques such as fluorescence [17]. In the present prospective observational study, we present the first application of in vivo RCM for the evaluation of the human skin microcirculation and simultaneously the histomorphological pattern on cellular levels in a temperature-dependant model. 2. Patients and methods 2.1. Patients
∗ Corresponding author. Tel.: +49 511 532 8865; fax: +49 511 532 8869. E-mail addresses: [email protected] (M.A. Altintas), [email protected] (M. Meyer-Marcotty), [email protected] (A.A. Altintas), [email protected] (M. Guggenheim), [email protected] (A. Gohritz), aust [email protected] (M.C. Aust), [email protected] (P.M. Vogt). 0895-6111/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.compmedimag.2009.04.011
20 healthy volunteers were randomly divided in two groups and subjected to the following procedures: • local heat stress by immersion in a water bath at 42 ± 3 ◦ C for a period of 120 s (HS-group; aged 21.8 ± 2.9 years, 4 female, 6 male);
M.A. Altintas et al. / Computerized Medical Imaging and Graphics 33 (2009) 532–536
• local cold stress by immersion in a water bath at 3 ± 1 ◦ C for a period of 120 s (CS-group; aged 23.7 ± 4.9 years, 5 female, 5 male). RCM was performed prior (control) and after local temperature stress on the previously marked area on the middle volar forearm which was covered with a water-impermeable sheet (OpSite foil, Smith and Nephew Germany, Hamburg, Germany) prior to immersion in the water bath. Written informed consent was obtained previous to enrolment in the study. All measurements were performed under controlled and standardized conditions at 22 ± 1 ◦ C and 50 ± 5% air humidity. In order not to confound histomorphological data, subjects with a history or evidence of diabetes, hypertension, and vascular disease were excluded. 2.2. Instrument
533
dow. These features provide both excellent tissue stabilization and imaging without applying pressure. Thus, the area of interest in the centre of the tissue ring is free of pressure in order to avoid the transmission into deeper layers and possibly on feeding and draining vessels. 2.3. Parameters The following parameters were assessed using RCM in a single area of interest in each subject prior and after local temperature stress: • Quantitative blood-cell flow per minute was established by counting in the capillary loops (see Fig. 2) of two papillae in each
The Vivascope1500 (Lucid Inc., Rochester, New York, USA, Fig. 1) is a specially constructed reflectance-mode confocal microscope for high resolution imaging of the human skin. Contrary to other confocal microscopes, this device is characterized by a GalliumArsenide laser source emitting in a long wavelength band at 830 nm. Thus it is in the “optical window” of the human skin and enables imaging up to a controlled depth of 350 m. The laser source is operating at a harmless energy of less than 30 mW at the skin surface and generates real-time imaging due to imaging rates of nine frames per second. High resolution imaging of the human epidermis and upper dermis occurs due to a lateral resolution of 0.4 m and a vertical resolution of 1.9 m. The field of vision of a single frame measures 500 m × 500 m. This microscope uses a specially constructed and patented magnetic metal tissue ring (Lucid Inc., Rochester, New York, USA) of 3 cm diameter with a glass win-
Fig. 1. Reflectance-mode confocal-laser-scanning microscopy (Vivascope1500; Lucid Inc., Rochester, New York, USA).
Fig. 2. Reflectance-mode confocal microscopy depicts the epidermal–dermal junction (a) and zoom of dermal papillae (b) in virtual horizontal sectioning. The basal layer circles (arrowhead) reflect strongly due to melanin content. The circles represent horizontal sections of dermal papillae and are surrounded by the spinous layer (star). The dark focus of the circles (ring) corresponds to the dermis and includes the luminae of capillary loops as black holes (arrows). In the focus of the black holes, transcapillary flow of single bright reflecting blood cells can be observed in realtime imaging. The contrast of confocal images is due to different naturally occurring refractive indexes of tissue structures, such as melanin, keratin, and cytoplasm.
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M.A. Altintas et al. / Computerized Medical Imaging and Graphics 33 (2009) 532–536
Fig. 3. En face confocal image at the level of the apical granular layer depicts in a field of 500 m × 500 m dark and large nucleated polygonal keratinocytic cells with granular cytoplasm arranged in a honeycomb pattern (arrows). The size of cells was measured in the apical granular layer using the image analysis program Image Tool.
volunteer by off-line analysis of four fields of view, which were digitally recorded for 30 s. • The capillary loop diameter was evaluated in digital images of the epidermal-dermal junction (see Fig. 2) using the image analysis program “Image Tool” (see Section 2.4). Two papillae were measured in four fields of view. • The granular cell size was assessed by evaluating confocal images of the apical plane of the granular layer with the focus showing dark nucleated cells (see Fig. 3). In each 10 fields of vision four cells were measured using the image analysis program “Image Tool”. • The basal layer thickness was assessed by evaluating images of the epidermal–dermal junction (see Fig. 2) using the same image analysis software; four measurements per papilla and 10 measurements per patient were taken. 2.4. Data and statistical analysis Image analyses were performed using a non-commercial image analysis program, “Image Tool”, Version 3.0 (UTHSCA, San Antonio, TX, USA). This software provides numerous image analysis tools, such as area or distance measurements (Figs. 3, 4 and 5). The results are presented in table form and therefore facilitate export to statistical software. The evaluated data were analyzed in blinded fashion by a statistician to determine the statistical significance. For statistical analyses, SPSS 16.0 for Windows was used. Analysis of distribution was performed using Kolmogorov–Smirnov test. Values of each parameter were averaged and expressed as mean ± SD. Independent-samples t-test was used, since the data fit a normal distribution. P-values of less than 0.05 were considered significant. 3. Results 3.1. HS-group Quantitative blood cell flow was 60.02 ± 3.11/min prior and increased significantly to 76.12 ± 2.97/min after local heat stress. The diameter of capillary loops was 9.10 ± 0.22 m (control) and increased to 11.04 ± 0.20 m after heating (P < 0.05). The granular
Fig. 4. Both blood cell flow, assessed by offline video analyses and capillary size, evaluated by confocal image analyses, increased significantly (*P < 0.05) after local heat stress, compared to the control.
cell size increased insignificantly to 749.8 ± 79.8 m2 , compared to the control at 732.9 ± 82.6 m2 . In addition, the basal layer thickness in controls was 14.7 ± 0.6 m and increased insignificantly to 15.0 ± 0.8 m in heated areas. 3.2. CS-group Blood cell flow was 60.63 ± 3.40/min in controls and decreased significantly to 40.62 ± 2.50/min after cold stress. The baseline for capillary size was 9.34 ± 0.26 m and decreased to 7.66 ± 0.26 m after cold stress (P < 0.05). The granular cell size was 736.8 ± 79.4 m2 in controls and decreased insignificantly after cold stress to 725.1 ± 73.1 m2 . The basal layer thickness decreased, however, insignificantly after cold stress to 14.3 ± 0.5 m compared to 14.9 ± 0.6 m (control). 4. Discussion Our results evaluated in vivo by RCM are in agreement with previous studies on temperature induced alterations in human skin microcirculation [11–14]. Recently, Ciplak et al. studied the vasodilatory response of skin microcirculation to local heating using laser Doppler imaging [12]. Using non-invasive near infrared spectrometer, Gomez et al. examined the dynamic tissue oxygen saturation [3]. However, these procedures enable neither the visualization of the vascular architecture, nor the transcapillary flow of the blood cells. Currently, RCM was validated in the burn care arena for characterizing and quantifying burn injuries, including burn depth determination, and in dermatology to evaluate various skin lesions suspected of malignancy [7,18–20]. To the best of
M.A. Altintas et al. / Computerized Medical Imaging and Graphics 33 (2009) 532–536
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human skin efficiently. In this respect, RCM may provide new diagnostic and/or therapy control options in the future. The relatively simple use, the non-invasive and high resolution character of this real-time imaging technique make it very attractive for studying the human skin physiology and pathology. We believe that, given the potential of confocal technique, it will extend its application to basic microcirculation research of the human skin. However, confocal microscopy is not without limitations. Currently this technique allows the acquisition of high resolution images up to a depth of 350 m only. Beyond this depth, however, image resolution is limited. Hence, only superficial vessels of the skin can be evaluated using this technique. Despite this fact, RCM is currently the only method that enables evaluation of the human skin microcirculation, including the histomorphology in vivo and in real time on cellular levels. Although the initial purchase costs of a confocal microscope might appear as drawback to its clinical applicability, we believe the ability to measure in vivo dynamic tissue changes in an atraumatic repetitive fashion is an unsurpassed advantage of this technique. Conflict of interest All authors disclose no conflicts of interest in terms of financial and personal relationships with other people or organisations that could inappropriately influence this work. Acknowledgement There were no funding sources for this study. Fig. 5. Using reflectance-mode confocal microscopy the evaluated single blood-cell flow and capillary size increased significantly (*P < 0.05) after local cold stress of the volar forearm compared to controls.
our knowledge, however, previous to our study, it was not used for the evaluation of dynamic, temperature-dependant changes of the human skin microcirculation and the associated histomorphological patterns. In a Laser–Doppler study, Christen et al. reported heating-induced local vasodilatation [15]. In accordance, using RCM and direct visualization of the dermal capillaries we observed a significant increase of the capillary size after local heat stress in all volunteers. Moreover, increased transcapillary flow of single blood cells was assessed by high resolution real-time imaging [16]. Vasoconstriction as the result of local cold stress on the human skin was previously reported as a part of local control, arising from sympathetic, sensory and autonomic nerves [21]. Based on RCM observations, we are not only able to confirm, but also to quantify this fact in vivo on a histomorphological level, as we assessed cold stress-dependant significant decrease of the capillary size. Moreover, in accordance with previous studies [14] we observed a concomitant decrease of single blood cell flow, induced by local cold stress. In this study, the area of interest was covered prior to contact with water in order not to confound histomorphological data. As expected, the observed histomorphological patterns reveal no significant differences prior and after immersion in water bath. However, we demonstrated that confocal technique enables not solely direct observation of dynamic microcirculatory processes, but also the evaluation of human skin histomorphology on cellular levels. In contrast to the study by Shen et al. [17], in our study we induced no damage using slightly milder temperature changes, while using a much simpler method. Various physiologic and pathophysiologic conditions are associated with functional and dynamic changes of microcirculation which affects directly the morphology of living skin. Thus evaluation of both microcirculation and the cellular morphology and integrity should be considered as irreplaceable to study the physiology and physiopathology of the
References [1] Ambrozy E, Waczulikova I, Willfort-Ehringer A, Ehringer H, Koppensteiner R, Gschwandtner ME. 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. [2] Belichenko VM, Korostishevskaya IM, Maximov VF, Shoshenko CA. Mitochondria and blood supply of chicken skeletal muscle fibers in ontogenesis. Microvasc Res 2004;68:265–72. [3] Gomez H, Torres A, Polanco P, Kim HK, Zenker S, Puyana JC, et al. Use of noninvasive NIRS during a vascular occlusion test to assess dynamic tissue O(2) saturation response. Intensive Care Med 2008;34:1600–7. [4] Setty BN, Betal SG. Microvascular endothelial cells express a phosphatidylserine receptor: a functionally active receptor for phosphatidylserine-positive erythrocytes. Blood 2008;111:905–14. [5] Szilagyi S. Pulse oximetry in the study of the viability of the intestines and the microcirculation in intestinal anastomoses (preliminary report). Orv Hetil 1994;135:1531–4. [6] Altintas MA, Altintas AA, Guggenheim M, Niederbichler AD, Knobloch K, Vogt PM. In vivo evaluation of histomorphological alterations in first-degree burn injuries by means of confocal-laser-scanning microscopy-more than “virtual histology?”. J Burn Care Res 2009;30:315–20. [7] Altintas MA, Altintas AA, Knobloch K, Guggenheim M, Zweifel CJ, Vogt PM. Differentiation of superficial-partial vs. deep-partial thickness burn injuries in vivo by confocal-laser-scanning microscopy. Burns 2009;35:80–6. [8] Gambichler T, Sauermann K, Altintas MA, Paech V, Kreuter A, Altmeyer P, et al. Effects of repeated sunbed exposures on the human skin, In vivo measurements with confocal microscopy. Photodermatol Photoimmunol Photomed 2004;20:27–32. [9] Gerger A, Koller S, Weger W, Richtig E, Kerl H, Samonigg H, et al. Sensitivity and specificity of confocal laser-scanning microscopy for in vivo diagnosis of malignant skin tumors. Cancer 2006;107:193–200. [10] Gonzalez S, Tannous Z. Real-time, in vivo confocal reflectance microscopy of basal cell carcinoma. J Am Acad Dermatol 2002;47:869–74. [11] Beed M, O’Connor MB, Kaur J, Mahajan RP, Moppett IK. Transient hyperaemic response to assess skin vascular reactivity: effects of heat and iontophoresed norepinephrine. Br J Anaesth 2009;102:205–9. [12] Ciplak M, Pasche A, Heim A, Haeberli C, Waeber B, Liaudet L, et al. The vasodilatory response of skin microcirculation to local heating is subject to desensitization. Microcirculation 2009;16:265–75. [13] Sokolnicki LA, Strom NA, Roberts SK, Roberts SK, Kingsley-Berg SA, Basu A, et al. Skin blood flow and nitric oxide during body heating in type 2 diabetes mellitus. J Appl Physiol 2009;106:566–70. [14] Li Z, Koman LA, Rosencrance E, Pollock DC, Smith BP, Strandhoy JW, et al. Effect of cooling on cutaneous microvascular adrenoceptors in vivo in the rabbit ear. J Orthop Res 1998;16:190–6.
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[15] Christen S, Delachaux A, Dischl B, Golay S, Liaudet L, Feihl F, et al. Dosedependent vasodilatory effects of acetylcholine and local warming on skin microcirculation. J Cardiovasc Pharmacol 2004;44:659–64. [16] Johnson JM, Yen TC, Zhao K, Kosiba WA. Sympathetic, sensory, and nonneuronal contributions to the cutaneous vasoconstrictor response to local cooling. Am J Physiol Heart Circ Physiol 2005;288:H1573–1579. [17] Shen Y, Liu P, Zhang A, Xu LX. Study on tumor microvasculature damage induced by alternate cooling and heating. Ann Biomed Eng 2008;36: 1409–19. [18] Gonzalez S, Gonzalez E, White WM, Rajadhyaksha M, Anderson RR. Allergic contact dermatitis: correlation of in vivo confocal imaging to routine histology. J Am Acad Dermatol 1999;40:708–13. [19] Rajadhyaksha M, Gonzalez S, Zavislan JM, Anderson RR, Webb RH. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. J Invest Dermatol 1999;113:293–303. [20] Gambichler T, Huyn J, Tomi NS, Moussa G, Moll C, Sommer A, et al. A comparative pilot study on ultraviolet-induced skin changes assessed by noninvasive imaging techniques in vivo. Photochem Photobiol 2006;82:1103–7. [21] Weiss T, Windthorst C, Weiss C, Kreuzer J, Bommer J, Kübler W. Acute effects of haemodialysis on cutaneous microcirculation in patients with peripheral arterial occlusive disease. Nephrol Dial Transplant 1998;13:2317–21. Mehmet Ali Altintas is currently a resident in the Department of Plastic-, Reconstructive and Hand surgery, Burn centre in the Medical School Hannover, Germany. He received his academic degree in medicine (MD) from the Ruhr University of Bochum, Germany in 2004. His research interests include, in vivo medical imaging and video processing, modulation of microcirculation, angiogenesis and burn surgery. Contact him at [email protected]. Max Meyer-Marcotty is currently working as a senior consultant in the Department of Plastic-, Reconstructive and Hand surgery, Burn centre in the Medical School Hannover, Germany. He received his academic degree in medicine (MD) at the Albert-Ludwigs-University Freiburg, Germany in 1997. He passed the German Speciality Boards in Plastic Surgery in Sep. 2005 and in Hand surgery in May 2008. The scientific interests of Dr. Meyer-Marcotty are reconstructive breast surgery, functional outcome of cryotherapy after wrist surgery. Ahmet Ali Altintas is currently working for the Department of Plastic and Reconstructive Surgery and Surgery of the Hand, Burn centre of the University of Witten,
Campus Cologne Merheim, Germany. His research interests include non-invasive imaging, image and video processing, content-based image retrieval, microcirculation and angiogenesis induction. Merlin Guggenheim is currently a consultant for Plastic and Reconstructive Surgery at the Division of Plastic Surgery, University Hospital Zurich, Switzerland. He completed Medical School at the University of Zurich, Switzerland in 1998 and his Doctorate in Medicine in 2000. He passed the European and Swiss Specialty Boards in Plastic Surgery in 2004 and 2006, respectively. Dr. Guggenheim’s research interests include oncoplastic surgery, burn surgery, bacterial biofilms, and non-invasive imaging. Andreas Gohritz, MD, is currently working at the Department of Plastic-, Hand and Reconstructive Surgery at the Hannover Medical School in Hannover / Germany. He studied medicine at the Ludwig-Maximilian-University in Munich/Germany with periods in Vienna/Austria, La Paz/Bolivia and Montpellier/France. He received his medical doctor degree at the Technical University in Munich. After three years in Munich (plastic surgery) and Bad Neustadt (hand surgery), he spent six months as a research fellow at the Sahlgrenska University, Department of Hand Surgery in Göteborg/Sweden with emphasis on functional upper extremity surgery in tetraplegic patients. He works in Hannover since 2005, his research interests include reconstruction in paralysis due to peripheral nerve and spinal cord injury, hand and microsurgery and the history of plastic surgery. Contact him at andreas [email protected]. Matthias Aust is currently a resident in the Department for Plastic-, Reconstructive and Hand surgery in the Medical School Hannover, Germany. He passed his medical exam and doctoral thesis with magna cum laude in 2004. He has worked in a number of renowned clinics for Plastic and Reconstructive Surgery in the US, Switzerland, South Africa, and Germany. His research interests in the field of anti aging. His experimental research focuses on skin regeneration and rejuvenation by percutaneous collagen induction therapy and other strategies in anti aging medicine. Prof. Vogt is currently the chairman of the Department for Plastic-, Reconstructive and Hand surgery of the Medical School Hannover, Germany.
Contents lists available at ScienceDirect
Computerized Medical Imaging and Graphics journal homepage: www.elsevier.com/locate/compmedimag
In vivo reflectance-mode confocal microscopy provides insights in human skin microcirculation and histomorphology M.A. Altintas a,∗ , M. Meyer-Marcotty a , A.A. Altintas b , M. Guggenheim c , A. Gohritz a , M.C. Aust a , P.M. Vogt a a b c
Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany Department of Plastic and Reconstructive Surgery, Cologne-Merheim University of Witten-Herdecke, Cologne, Germany Division of Plastic and Reconstructive Surgery, Burn Centre, Department of Surgery, University Hospital Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 11 January 2009 Received in revised form 17 April 2009 Accepted 20 April 2009 Keywords: Non-invasive imaging High resolution imaging Vasoconstriction Vasodilatation Blood flow
a b s t r a c t Purpose: Various approaches are used to study microcirculation, however, no modality evaluates microcirculation and histomorphology on cellular levels. We hypothesized that reflectance-mode confocal microscopy (RCM) enables simultaneous evaluation in vivo of both microcirculation and histomorphology. Principals: The forearm of 20 volunteers was exposed to either local heat stress (HS-group), or to local cold stress (CS-group). RCM was performed prior and after temperature stress to evaluate quantitative blood-cell flow, capillary loop diameter, granular cell size, and basal layer thickness. Results: In the HS-group, we observed significant increase in capillary loop diameter and increased bloodcell flow after heat stress. In the CS-group, significant decreases of capillary loop diameter and in bloodcell flow were determined following cold stress. Granular cell size and basal layer thickness differed insignificantly prior and after local temperature stress. Conclusions: RCM provides real-time and in vivo high resolution imaging of temperature-dependent changes in the human skin microcirculation and histomorphology on cellular levels. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction In the past years, various technical developments were evaluated to observe the human skin microcirculation in vivo, such as Laser Doppler Flowmetry [1], hydrogen clearance [2], near infrared spectroscopy [3], hemoglobin oxygenation [4] and pulsoxymetry [5]. However, none of these methods can sufficiently evaluate the dynamic microcirculation and simultaneously elucidate the cellular morphology and integrity. However, this information should be considered as irreplaceable to study the physiology and physiopathology of the skin efficiently. Thus, a high resolution imaging technique to visualize the human skin microcirculation and the associated cellular morphology would be novel in microcirculation research. Reflectance-mode confocal microscopy (RCM) enables real-time and in vivo high resolution imaging of the human skin and has been used in numerous studies in dermatology and the
burn care arena [6–10]. For correlation, confocal images were compared to histological sectioning and a high sensitivity of 82.9% and specificity of 95.7% was found [9,10]. The dynamic effects of temperature changes on the human skin microcirculation are well investigated using non-invasive approaches and also well described in the actual literature [11–16]. Temperature-dependent microcirculation changes have been studied, but only at temperatures that damage the skin, using more complex techniques such as fluorescence [17]. In the present prospective observational study, we present the first application of in vivo RCM for the evaluation of the human skin microcirculation and simultaneously the histomorphological pattern on cellular levels in a temperature-dependant model. 2. Patients and methods 2.1. Patients
∗ Corresponding author. Tel.: +49 511 532 8865; fax: +49 511 532 8869. E-mail addresses: [email protected] (M.A. Altintas), [email protected] (M. Meyer-Marcotty), [email protected] (A.A. Altintas), [email protected] (M. Guggenheim), [email protected] (A. Gohritz), aust [email protected] (M.C. Aust), [email protected] (P.M. Vogt). 0895-6111/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.compmedimag.2009.04.011
20 healthy volunteers were randomly divided in two groups and subjected to the following procedures: • local heat stress by immersion in a water bath at 42 ± 3 ◦ C for a period of 120 s (HS-group; aged 21.8 ± 2.9 years, 4 female, 6 male);
M.A. Altintas et al. / Computerized Medical Imaging and Graphics 33 (2009) 532–536
• local cold stress by immersion in a water bath at 3 ± 1 ◦ C for a period of 120 s (CS-group; aged 23.7 ± 4.9 years, 5 female, 5 male). RCM was performed prior (control) and after local temperature stress on the previously marked area on the middle volar forearm which was covered with a water-impermeable sheet (OpSite foil, Smith and Nephew Germany, Hamburg, Germany) prior to immersion in the water bath. Written informed consent was obtained previous to enrolment in the study. All measurements were performed under controlled and standardized conditions at 22 ± 1 ◦ C and 50 ± 5% air humidity. In order not to confound histomorphological data, subjects with a history or evidence of diabetes, hypertension, and vascular disease were excluded. 2.2. Instrument
533
dow. These features provide both excellent tissue stabilization and imaging without applying pressure. Thus, the area of interest in the centre of the tissue ring is free of pressure in order to avoid the transmission into deeper layers and possibly on feeding and draining vessels. 2.3. Parameters The following parameters were assessed using RCM in a single area of interest in each subject prior and after local temperature stress: • Quantitative blood-cell flow per minute was established by counting in the capillary loops (see Fig. 2) of two papillae in each
The Vivascope1500 (Lucid Inc., Rochester, New York, USA, Fig. 1) is a specially constructed reflectance-mode confocal microscope for high resolution imaging of the human skin. Contrary to other confocal microscopes, this device is characterized by a GalliumArsenide laser source emitting in a long wavelength band at 830 nm. Thus it is in the “optical window” of the human skin and enables imaging up to a controlled depth of 350 m. The laser source is operating at a harmless energy of less than 30 mW at the skin surface and generates real-time imaging due to imaging rates of nine frames per second. High resolution imaging of the human epidermis and upper dermis occurs due to a lateral resolution of 0.4 m and a vertical resolution of 1.9 m. The field of vision of a single frame measures 500 m × 500 m. This microscope uses a specially constructed and patented magnetic metal tissue ring (Lucid Inc., Rochester, New York, USA) of 3 cm diameter with a glass win-
Fig. 1. Reflectance-mode confocal-laser-scanning microscopy (Vivascope1500; Lucid Inc., Rochester, New York, USA).
Fig. 2. Reflectance-mode confocal microscopy depicts the epidermal–dermal junction (a) and zoom of dermal papillae (b) in virtual horizontal sectioning. The basal layer circles (arrowhead) reflect strongly due to melanin content. The circles represent horizontal sections of dermal papillae and are surrounded by the spinous layer (star). The dark focus of the circles (ring) corresponds to the dermis and includes the luminae of capillary loops as black holes (arrows). In the focus of the black holes, transcapillary flow of single bright reflecting blood cells can be observed in realtime imaging. The contrast of confocal images is due to different naturally occurring refractive indexes of tissue structures, such as melanin, keratin, and cytoplasm.
534
M.A. Altintas et al. / Computerized Medical Imaging and Graphics 33 (2009) 532–536
Fig. 3. En face confocal image at the level of the apical granular layer depicts in a field of 500 m × 500 m dark and large nucleated polygonal keratinocytic cells with granular cytoplasm arranged in a honeycomb pattern (arrows). The size of cells was measured in the apical granular layer using the image analysis program Image Tool.
volunteer by off-line analysis of four fields of view, which were digitally recorded for 30 s. • The capillary loop diameter was evaluated in digital images of the epidermal-dermal junction (see Fig. 2) using the image analysis program “Image Tool” (see Section 2.4). Two papillae were measured in four fields of view. • The granular cell size was assessed by evaluating confocal images of the apical plane of the granular layer with the focus showing dark nucleated cells (see Fig. 3). In each 10 fields of vision four cells were measured using the image analysis program “Image Tool”. • The basal layer thickness was assessed by evaluating images of the epidermal–dermal junction (see Fig. 2) using the same image analysis software; four measurements per papilla and 10 measurements per patient were taken. 2.4. Data and statistical analysis Image analyses were performed using a non-commercial image analysis program, “Image Tool”, Version 3.0 (UTHSCA, San Antonio, TX, USA). This software provides numerous image analysis tools, such as area or distance measurements (Figs. 3, 4 and 5). The results are presented in table form and therefore facilitate export to statistical software. The evaluated data were analyzed in blinded fashion by a statistician to determine the statistical significance. For statistical analyses, SPSS 16.0 for Windows was used. Analysis of distribution was performed using Kolmogorov–Smirnov test. Values of each parameter were averaged and expressed as mean ± SD. Independent-samples t-test was used, since the data fit a normal distribution. P-values of less than 0.05 were considered significant. 3. Results 3.1. HS-group Quantitative blood cell flow was 60.02 ± 3.11/min prior and increased significantly to 76.12 ± 2.97/min after local heat stress. The diameter of capillary loops was 9.10 ± 0.22 m (control) and increased to 11.04 ± 0.20 m after heating (P < 0.05). The granular
Fig. 4. Both blood cell flow, assessed by offline video analyses and capillary size, evaluated by confocal image analyses, increased significantly (*P < 0.05) after local heat stress, compared to the control.
cell size increased insignificantly to 749.8 ± 79.8 m2 , compared to the control at 732.9 ± 82.6 m2 . In addition, the basal layer thickness in controls was 14.7 ± 0.6 m and increased insignificantly to 15.0 ± 0.8 m in heated areas. 3.2. CS-group Blood cell flow was 60.63 ± 3.40/min in controls and decreased significantly to 40.62 ± 2.50/min after cold stress. The baseline for capillary size was 9.34 ± 0.26 m and decreased to 7.66 ± 0.26 m after cold stress (P < 0.05). The granular cell size was 736.8 ± 79.4 m2 in controls and decreased insignificantly after cold stress to 725.1 ± 73.1 m2 . The basal layer thickness decreased, however, insignificantly after cold stress to 14.3 ± 0.5 m compared to 14.9 ± 0.6 m (control). 4. Discussion Our results evaluated in vivo by RCM are in agreement with previous studies on temperature induced alterations in human skin microcirculation [11–14]. Recently, Ciplak et al. studied the vasodilatory response of skin microcirculation to local heating using laser Doppler imaging [12]. Using non-invasive near infrared spectrometer, Gomez et al. examined the dynamic tissue oxygen saturation [3]. However, these procedures enable neither the visualization of the vascular architecture, nor the transcapillary flow of the blood cells. Currently, RCM was validated in the burn care arena for characterizing and quantifying burn injuries, including burn depth determination, and in dermatology to evaluate various skin lesions suspected of malignancy [7,18–20]. To the best of
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human skin efficiently. In this respect, RCM may provide new diagnostic and/or therapy control options in the future. The relatively simple use, the non-invasive and high resolution character of this real-time imaging technique make it very attractive for studying the human skin physiology and pathology. We believe that, given the potential of confocal technique, it will extend its application to basic microcirculation research of the human skin. However, confocal microscopy is not without limitations. Currently this technique allows the acquisition of high resolution images up to a depth of 350 m only. Beyond this depth, however, image resolution is limited. Hence, only superficial vessels of the skin can be evaluated using this technique. Despite this fact, RCM is currently the only method that enables evaluation of the human skin microcirculation, including the histomorphology in vivo and in real time on cellular levels. Although the initial purchase costs of a confocal microscope might appear as drawback to its clinical applicability, we believe the ability to measure in vivo dynamic tissue changes in an atraumatic repetitive fashion is an unsurpassed advantage of this technique. Conflict of interest All authors disclose no conflicts of interest in terms of financial and personal relationships with other people or organisations that could inappropriately influence this work. Acknowledgement There were no funding sources for this study. Fig. 5. Using reflectance-mode confocal microscopy the evaluated single blood-cell flow and capillary size increased significantly (*P < 0.05) after local cold stress of the volar forearm compared to controls.
our knowledge, however, previous to our study, it was not used for the evaluation of dynamic, temperature-dependant changes of the human skin microcirculation and the associated histomorphological patterns. In a Laser–Doppler study, Christen et al. reported heating-induced local vasodilatation [15]. In accordance, using RCM and direct visualization of the dermal capillaries we observed a significant increase of the capillary size after local heat stress in all volunteers. Moreover, increased transcapillary flow of single blood cells was assessed by high resolution real-time imaging [16]. Vasoconstriction as the result of local cold stress on the human skin was previously reported as a part of local control, arising from sympathetic, sensory and autonomic nerves [21]. Based on RCM observations, we are not only able to confirm, but also to quantify this fact in vivo on a histomorphological level, as we assessed cold stress-dependant significant decrease of the capillary size. Moreover, in accordance with previous studies [14] we observed a concomitant decrease of single blood cell flow, induced by local cold stress. In this study, the area of interest was covered prior to contact with water in order not to confound histomorphological data. As expected, the observed histomorphological patterns reveal no significant differences prior and after immersion in water bath. However, we demonstrated that confocal technique enables not solely direct observation of dynamic microcirculatory processes, but also the evaluation of human skin histomorphology on cellular levels. In contrast to the study by Shen et al. [17], in our study we induced no damage using slightly milder temperature changes, while using a much simpler method. Various physiologic and pathophysiologic conditions are associated with functional and dynamic changes of microcirculation which affects directly the morphology of living skin. Thus evaluation of both microcirculation and the cellular morphology and integrity should be considered as irreplaceable to study the physiology and physiopathology of the
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[15] Christen S, Delachaux A, Dischl B, Golay S, Liaudet L, Feihl F, et al. Dosedependent vasodilatory effects of acetylcholine and local warming on skin microcirculation. J Cardiovasc Pharmacol 2004;44:659–64. [16] Johnson JM, Yen TC, Zhao K, Kosiba WA. Sympathetic, sensory, and nonneuronal contributions to the cutaneous vasoconstrictor response to local cooling. Am J Physiol Heart Circ Physiol 2005;288:H1573–1579. [17] Shen Y, Liu P, Zhang A, Xu LX. Study on tumor microvasculature damage induced by alternate cooling and heating. Ann Biomed Eng 2008;36: 1409–19. [18] Gonzalez S, Gonzalez E, White WM, Rajadhyaksha M, Anderson RR. Allergic contact dermatitis: correlation of in vivo confocal imaging to routine histology. J Am Acad Dermatol 1999;40:708–13. [19] Rajadhyaksha M, Gonzalez S, Zavislan JM, Anderson RR, Webb RH. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. J Invest Dermatol 1999;113:293–303. [20] Gambichler T, Huyn J, Tomi NS, Moussa G, Moll C, Sommer A, et al. A comparative pilot study on ultraviolet-induced skin changes assessed by noninvasive imaging techniques in vivo. Photochem Photobiol 2006;82:1103–7. [21] Weiss T, Windthorst C, Weiss C, Kreuzer J, Bommer J, Kübler W. Acute effects of haemodialysis on cutaneous microcirculation in patients with peripheral arterial occlusive disease. Nephrol Dial Transplant 1998;13:2317–21. Mehmet Ali Altintas is currently a resident in the Department of Plastic-, Reconstructive and Hand surgery, Burn centre in the Medical School Hannover, Germany. He received his academic degree in medicine (MD) from the Ruhr University of Bochum, Germany in 2004. His research interests include, in vivo medical imaging and video processing, modulation of microcirculation, angiogenesis and burn surgery. Contact him at [email protected]. Max Meyer-Marcotty is currently working as a senior consultant in the Department of Plastic-, Reconstructive and Hand surgery, Burn centre in the Medical School Hannover, Germany. He received his academic degree in medicine (MD) at the Albert-Ludwigs-University Freiburg, Germany in 1997. He passed the German Speciality Boards in Plastic Surgery in Sep. 2005 and in Hand surgery in May 2008. The scientific interests of Dr. Meyer-Marcotty are reconstructive breast surgery, functional outcome of cryotherapy after wrist surgery. Ahmet Ali Altintas is currently working for the Department of Plastic and Reconstructive Surgery and Surgery of the Hand, Burn centre of the University of Witten,
Campus Cologne Merheim, Germany. His research interests include non-invasive imaging, image and video processing, content-based image retrieval, microcirculation and angiogenesis induction. Merlin Guggenheim is currently a consultant for Plastic and Reconstructive Surgery at the Division of Plastic Surgery, University Hospital Zurich, Switzerland. He completed Medical School at the University of Zurich, Switzerland in 1998 and his Doctorate in Medicine in 2000. He passed the European and Swiss Specialty Boards in Plastic Surgery in 2004 and 2006, respectively. Dr. Guggenheim’s research interests include oncoplastic surgery, burn surgery, bacterial biofilms, and non-invasive imaging. Andreas Gohritz, MD, is currently working at the Department of Plastic-, Hand and Reconstructive Surgery at the Hannover Medical School in Hannover / Germany. He studied medicine at the Ludwig-Maximilian-University in Munich/Germany with periods in Vienna/Austria, La Paz/Bolivia and Montpellier/France. He received his medical doctor degree at the Technical University in Munich. After three years in Munich (plastic surgery) and Bad Neustadt (hand surgery), he spent six months as a research fellow at the Sahlgrenska University, Department of Hand Surgery in Göteborg/Sweden with emphasis on functional upper extremity surgery in tetraplegic patients. He works in Hannover since 2005, his research interests include reconstruction in paralysis due to peripheral nerve and spinal cord injury, hand and microsurgery and the history of plastic surgery. Contact him at andreas [email protected]. Matthias Aust is currently a resident in the Department for Plastic-, Reconstructive and Hand surgery in the Medical School Hannover, Germany. He passed his medical exam and doctoral thesis with magna cum laude in 2004. He has worked in a number of renowned clinics for Plastic and Reconstructive Surgery in the US, Switzerland, South Africa, and Germany. His research interests in the field of anti aging. His experimental research focuses on skin regeneration and rejuvenation by percutaneous collagen induction therapy and other strategies in anti aging medicine. Prof. Vogt is currently the chairman of the Department for Plastic-, Reconstructive and Hand surgery of the Medical School Hannover, Germany.