Laser Doppler imaging and capillary microscopy in ischemic ulcers

Transcription

1 Atherosclerosis 142 (1999) Laser Doppler imaging and capillary microscopy in ischemic ulcers Michael E. Gschwandtner a, Ewald Ambrózy a, Barbara Schneider b, Sonja Fasching a, Andrea Willfort a, Herbert Ehringer a, * a Department of Medical Angiology, Allgemeines Krankenhaus Wien Währinger Gürtel 18-20, A-1090 Vienna, Austria b Department of Medical Statistics, Allgemeines Krankenhaus Wien Währinger Gürtel 18-20, A-1090 Vienna, Austria Received 16 December 1997; received in revised form 8 July 1998; accepted 28 July 1998 Abstract The local distribution of laser Doppler flux (mainly thermoregulatory perfusion) and capillary density (nutritive circulation) within 25 ischemic leg ulcers and their adjacent skin were investigated. For this purpose the technique of laser Doppler imaging and capillary microscopy were applied. In each ulcer a non granulation tissue area (NGTA), a granulation tissue area (GTA) and in adjacent skin a skin area (SA) were defined. In these areas the average laser Doppler area flux (arbitrary units, AU) and the number of capillaries/mm 2 were determined for each patient. The mean S.D. of laser Doppler area fluxes were: NGTA , GTA and SA AU, respectively. The differences between GTA and NGTA or SA was statistically significant (p 0.001, each) The mean S.D. of capillary densities were as follows: NGTA: , GTA and SA capillaries/mm 2, respectively. The following differences were statistically significant: NGTA versus GTA (p 0.01) and SA versus NGTA or GTA (p 0.001, each). In conclusion following characteristics of the three areas can be described: In NGTA low laser Doppler area flux is combined with very low capillary density (ulcer area without healing). In GTA the highest laser Doppler area flux of all three areas and an intermediate capillary density (wound healing) is found. In SA an intermediate laser Doppler area flux is associated with the highest capillary density of all three areas with the healing process nearly completed and no granulation tissue Elsevier Science Ireland Ltd. All rights reserved. Keywords: Laser Doppler imager; Laser Doppler flux; Capillary microscopy; Ischemic ulcer; Granulation tissue 1. Introduction * Corresponding author. Tel.: ; fax: Skin microcirculation is known to consist of two functionally different networks: (a) the superficial, nutritive; and (b) the deeper, mainly thermoregulatory vascular bed [1]. For examination of each network, different techniques have been established [2]. The nutritional network of the skin can be examined by capillary microscopy, which has a short penetration depth of a few microns; the number of visible capillaries in the skin correlates with the degree of compensation of peripheral arterial occlusive disease (PAOD). In chronic critical limb ischemia the number of capillaries is reduced and in severe cases even avascular fields are found [3 6]. A laser Doppler can be used for investigation of the mainly thermoregulatory bed, which is located in deeper layers of the skin [1]. Nutritive circulation is also partly determined by this method. If this technique is combined with capillary microscopy, the thermoregulatory portion of the laser Doppler signal can be estimated [7]. Signals measured with the laser Doppler are calculated values of the product of blood cell average speed and concentration [8]. Directional effects are usually not considered, except in flux measurements of single vessels [8]. In 1993 a further development of this technique was introduced: the laser Doppler imager (LDI) [9]. This device makes it possible, to map the local distribution of the laser Doppler flux without /99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S (98)

2 226 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) direct contact, thus producing a two-dimensional, color-coded image. In a recent study the laser Doppler flux distribution within ischemic ulcers and their adjacent skin was shown by our group. In this study, an inhomogeneous laser Doppler flux distribution within ulcers was found. Within the ulcers, areas with granulation tissue showed higher fluxes than did areas without granulation tissue [10]. The aim of the present study was to examine microcirculation within ischemic ulcers in more detail and to compare it with the adjacent skin. For this purpose the following questions were examined: How is the laser Doppler flux distributed in the ischemic ulcer in regard to the location of the granulation tissue? How is the laser Doppler flux distributed in the ulcer in regard to that of the adjacent skin? How is the capillary density distributed in the ischemic ulcer in regard to the location of the granulation tissue? How is the capillary density distributed in the ulcer in regard to that of the adjacent skin? How is the laser Doppler flux distributed in regard to the corresponding capillary density within the ulcer and in the adjacent skin 2. Patients and methods 2.1. Patients Twenty-five patients suffering from ischemic ulcers of the legs were studied. The patients comprised 11 male (44%) and 14 (56%) female Caucasians at the average age of years, the average body weight of kg and the average height of m (mean S.D.). Ten (40%) diabetics were included in this study. Peripheral arterial pressures (mmhg) were measured by ankle and arm cuffs using the Doppler ultrasound technique (Multi Dop L; NBN, Sipplingen, Germany). Toe pressures (mmhg) were determined using toe cuffs and photoplethysmographic sensors (Meda Sonics PA 13 Photo Pulse Adaptor; CA, USA) [11]. Mean S.D. ankle pressures of the ischemic legs investigated were (anterior tibial artery) and mmhg (posterior tibial artery). In 11 patients toe pressures were too low to be measured, in the remaining 14 patients mean S.D. toe pressure was mmhg. In each patient location and extent of the underlying peripheral arterial occlusive disease was proven by angiography. All patients examined met the criteria of chronic critical leg ischemia according to the European Consensus Document [12]. The arterial ulcers examined in this study were located as follows: 15 (60%) on forefeet, seven (28%) on toes and three (12%) on ankles or heels. The average ulcer diameter was mm and the mean history, since the development of the ischemic lesion, was months (mean S.D.) Measurements Ischemic ulcers with clean surfaces were included only. In addition, areas with and without granulation tissue had to be visible by the naked eye. In some cases, mechanical debridement was necessary to remove all non-viable-material, to avoid occlusion of the laser beam or the incident light of capillary microscopy. In these patients measurements were started after an interval of at least 24 h following the most recent cleaning manipulation, to avoid any influence on the measurements [13,14]. In addition, patients had to acclimatize for at least 30 min at an average room temperature of C. The entire investigation was performed with the patients in supine position (see Section 4). Examinations were carried out according to the following protocol: (1) First of all a macroscopic photograph was taken (Polaroid, Sonar One Step SX 70, Cambridge, MA, USA). Depending on the macroscopic appearance the following three regions of interest (ROI) were defined and marked on the photograph by frames (Fig. 1a; macroscopic photograph on the left). Within the ulcer two areas were defined: a pale area representing an area without granulation tissue (non granulation tissue area, NGTA) (Fig. 1a; area A) and a more erythematous area than the other ulcer area, representing an area with granulation tissue (granulation tissue area, GTA) (Fig. 1a; area B). These two ischemic ulcer areas, NGTA and GTA, were characterized earlier by histologic findings (see Section 4). As the third region, an adjacent skin area (skin area, SA) (Fig. 1a; area C), was defined. The latter ROI had to be located between 3 and 15 mm from the ulcer s border. To avoid bias, these regions had to be defined before performing further examination. (2) The second step was to generate a laser Doppler image by means of the laser Doppler imager (LDI) (PIM 1.0, Lisca Development AB, Sweden). An LDI is a noninvasive medical device, that creates two-dimensional, color coded images of tissue perfusion. A laser beam of a LDI (wavelength: 632 nm, power: 1 mw) scans the investigated tissue. In the tissue the laser beam becomes Doppler shifted by inference with moving blood cells. The Doppler shifted and backscattered beam is picked up by a photo detector. There the beam is processed and mixed, to form a photocurrent. The photocurrent scales linearly with tissue perfusion, defined as the product of blood cell average speed and concentration. The output signal is displayed in the form of a color-coded image on a computer monitor (see Fig. 1b; on the right): a poor perfusion is expressed by blue, whereas higher flux is coded by increasing quantiles of green, yellow and red [9]. Within the laser Doppler image the areas of interest were defined in agreement with the macroscopic pho-

3 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) Fig. 1. Macroscopic photograph of an ischemic ulcer (a, left side) with the corresponding laser Doppler image (b, right side). The three regions of interest are marked with capital letters: A, ischemic ulcer non granulation tissue area; B, ischemic ulcer granulation tissue area; C, skin area. Fig. 2. Corresponding capillary microscopy of the three areas of the same ischemic ulcer as shown in Fig. 1: (A) ischemic ulcer non granulation tissue area; (B) ischemic ulcer granulation tissue area; (C) skin area.

4 228 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) tograph: areas corresponding with NGTA, GTA and SA (Fig. 1b, area A, B and C on the laser Doppler image on the right). To enable a similar placement of the ROI specific landmarks, such as the hyperemic border of the ulcer or other eminent structures, were used as an orientation. By the use of this procedure the investigated areas were intended to correspond to the macroscopic appearance in terms of position and size. In our example (Fig. 1b, right side) NGTA (area A) was coded blue indicating poorest flux in the laser Doppler image (compare legend on the right). In contrast GTA (area B) was coded green, yellow and red. This region represented the highest laser Doppler flux within the image. SA (area C), close to the ulcer s border, was coded mainly blue and green, showing an intermediate flux. The more distant skin appeared homogeneously blue representing low ischemic resting flux. Within these three ROI marked, the mean laser Doppler area flux (referred to hereafter as the flux ) was calculated by the use of the incorporated software program of the laser Doppler imager and expressed in arbitrary units (AU). (3) Capillary microscopy was performed as the third step using a similar videomicroscopic system as described earlier [7]. Capillaries were visualized using an incident-light excited microscope (Leitz Wetzlar, Germany), also applicable in standard fluorescence techniques. The light-filter-lens system (Ploemopak 2.3, Leitz, Wetzlar, Germany) consisted of a mercury vapor lamp as light source, a heat filter (UVR 380 nm, Leitz Wetzlar, Germany) a red-suppression filter, a field diaphragm, a filter system including a pole cube filter and a dichroic beam-splitting mirror. A 4-fold objective (Pl Fl 4/0.14, Leitz, Wetzlar, Germany) yielded a 300-fold final magnification on the monitor. To achieve a better contrast, a drop of paraffin oil was applied upon the investigated area. The microscope was mounted on a firm stand (Foba, Wettswil, Switzerland), which permitted three-dimensional adjustment of the objectives to the investigated area. Via a sensitive digital videocamera (Grundig FA 87 digital, Fürth, Germany), pictures were visualized on a television monitor (Sony Trinitron, Tokyo, Japan). All examinations were stored on a video tape, using a Super-VHS videocassette recorder (Panasonic NV-FS 200 HQ, Matsushita Electric Industrial Co., Ltd., Osaka, Japan). Once chosen for further evaluation microscopic patterns were stored on a photograph with the 45-fold final magnification, obtained by the use of a color video printer, Mavigraph (Sony, Tokyo, Japan). Capillary microscopy was carried out within the whole area comparable with the regions marked on the macroscopic photograph. In order to avoid getting the wrong number of capillaries due to an impaired transparency, the entire examination was looked through on a video tape. Thereby the area of the obviously best transparency was chosen to be representative for the capillary microscopic evaluation in NGTA, GTA and SA. Capillary density, expressed as the number of capillary loops in a selected visual field (i.e. per mm 2 ), was the basis of further evaluation. As shown in Fig. 2, NGTA (Fig. 2A) was characterized by NGTA capillary pattern: The dark area in the middle left represented microscopically small clumped material. On the lower right, an avascular area was found (corresponding with stage 6 of Fagrell s classification as described for the skin of the dorsum of the ischemic foot [4]). GTA capillary pattern (Fig. 2B) was characterized by a reduction of visible capillaries, which were not sharply outlined due to microedema (corresponding with stages 3 and 5 of Fagrell s classification of the skin). In the surrounding ischemic skin, in SA (Fig. 2C), capillary stages 2 and 3 according to Fagrell s classification were found: a moderate reduction of slightly dilated capillaries and a slight capillary edema Statistical analysis Flux and the capillary density values in each of the three ROI, as described above, form the basis for further statistical analysis. Distribution of the average flux and capillary density of each of the three ROI across patients were described using mean S.D. for the pooled sample. To detect differences between NGTA, GTA and SA, the paired t-test or the nonparametric sign rank test, as appropriate, was used. All p-values were two sided. A p-value less than 0.05 was considered statistically significant. To show association between flux and capillary density values Spearman s correlation coefficient was used. For data analysis the SAS software package was used. 3. Results 3.1. Laser Doppler area flux distribution in ischemic ulcers and adjacent skin In Fig. 3 the individual values of flux in each of the three ROI (NGTA, GTA, SA) of each of the 25 patients is connected by lines. Thus, the individual values of each patient in the three regions of interest can easily be followed. In addition, mean S.D. of flux is given for each region. Mean GTA-flux was highest, followed by mean NGTA- and mean SA-flux. In detail NGTA-flux varied from 0 (2/25) to 9.17 AU (1/25) resulting in a MEAN SD of AU. In GTA flux ranged from 0.22 (1/25) to 7.76 AU (1/25), resulting in a mean S.D. of AU. In SA flux ranged from 0.29 AU (2/25) to 3.2 AU (1/25), resulting in a

5 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) mean S.D. of AU. Flux in GTA was higher than that in NGTA or in SA (p each). In the majority of patients flux in NGTA was lower than in SA (17/25 patients). Due to the three patients with extraordinary high flux-values in NGTA (patients marked by 1, 2 and 3 in Fig. 3; see Section 4), mean S.D. was numerically, but not statistically higher in NGTA than in SA (p 0.05) Capillary density in ischemic ulcers and adjacent skin In Fig. 4 the individual capillary densities of the 3 ROI (NGTA, GTA and SA) of each of the 25 patients is connected by lines. Thus, the individual values of each patient in the three regions can easily be seen and mean S.D. of capillary densities for the three ROI is given. In contrast to distribution of flux, capillary density was highest in SA followed by GTA and NGTA. In detail, in NGTA the capillary density varied from 0 (22/25 patients) to 10 capillaries/mm 2, resulting in a mean S.D. of capillaries/mm 2. In NGTA only in 3/25 patients capillaries were visible. In GTA capillary density ranged from 0 (3/25) to 40 capillaries/mm 2 (1/25), resulting in a mean S.D. of capillaries/ mm 2.In23/25 patients capillary density was higher in SA than in GTA. One patient had an extraordinary high capillary density in GTA (40 capillaries/mm 2 in GTA). In SA the capillary density ranged from 4 (1/25) to 30 capillaries/mm 2 (2/25), resulting in a mean S.D. of capillaries/mm 2. Differences between all ROI were statistically significant (NGTA versus GTA: p 0.01; GTA versus SA: p and NGTA versus SA: p 0.001) Correlation between laser Doppler area flux and capillary density No significant difference of the correlation coefficient from 0 between flux and capillary density was detected. 4. Discussion To our knowledge, this is the first study, in which a combined evaluation of the capillary density and flux within ischemic ulcers NGTA and GTA is described. Furthermore, the findings within ulcers are compared with those in adjacent skin regions. The synoptic interpretation of capillary density on the one hand and flux on the other hand, could support the following view: In NGTA only a few visible capillaries were combined with low flux. NGTA capillary pattern is characterized by microscopically small, clumped necrotic material, avascular areas or areas of nearly no capillaries, respectively (Fig. 2A). These capillary findings could agree with the lack of a healing process, because avascular areas are also found in severe skin ischemia being at high risk for the development of necrosis [4,6]. Flux, on the other hand, was low, but not zero and might therefore give dependent on its level the basis for the later development of Fig. 3. Individual values of the average laser Doppler area flux of the ischemic ulcer non granulation tissue area, of the ischemic ulcer granulation tissue area and of the skin area are shown; the individual values of the three regions of interest (ROI) are connected by lines. In the lower part mean S.D. are given.

6 230 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) Fig. 4. Individual values of the capillary density of the ischemic ulcer non granulation tissue area, of the ischemic ulcer granulation tissue area and of the skin area are shown; the individual values of the three regions of interest (ROI) are connected by lines. In the lower part mean S.D. are given. granulation tissue or healing, respectively. The flux value required, which is necessary to initiate a healing process in not known until now. In GTA of the ischemic ulcer a significantly higher capillary density than in NGTA was combined with the highest flux measured. GTA capillary pattern was characterized by a low capillary density-less than in the ischemic skin near the ulcer; the capillaries are embedded in edema, and therefore they appear indistinct (Fig. 2B). In pronounced ischemia of the skin a low capillary density and edema can be seen, too [4]. The high flux obviously accompanies the active healing process in granulation tissue. In SA the relatively highest capillary density of the three ROI is combined with a rather low flux. The capillary density of the ischemic skin near the ulcer was reduced in comparison with normals, however (Fig. 2C; capillaries/mm 2 versus capillaries/mm 2 on the dorsum of the foot [7,15]); the capillary microscopic findings in SA near the ischemic ulcer are comparable with those of ischemic skin described by Fagrell: dilated capillaries and edema [4]. On the basis of this interpretation the fugitive patients of ischemic ulcer flux might be looked at. The three patients with an unexpected high flux in NGTA (patients 1, 2 and 3 in Fig. 3) might represent a very initial stage of wound healing preceding the development of visible granulation tissue: high flux with even some visible capillaries in one of them. In contrast to that, a very low flux in NGTA and GTA might represent patients with a poor wound healing prognosis; an interpretation, which of course requires a long time observation. It might be argued, that the three ROI were subjectively chosen by the investigators, and that this influenced the results. However, we tried to define the three areas before performing the study. The two ulcer areas as in Fig. 1a show different macroscopic appearances: one is pale, whereas the other is erythematous. The underlying histological findings correlated to such particular areas, which were examined earlier: In pale areas no granulation tissue, but necrotic material infiltrated by a few leukocytes and fibrin were microscopically observed. We named them therefore non granulation tissue areas (NGTA). In erythematous areas, tissues full of capillaries, the so called granulation tissues, and some leukocytes were found. We named them therefore granulation tissue areas (GTA). In the present study

7 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) no histologic examinations were done. However, one should suppose, that NGTA and GTA investigated in this study, corresponded with the histologic findings mentioned above. The macroscopic differences of NGTA and GTA obviously correspond with different pathophysiologic functions, because ulcer healing is known, to initiate from GTA. In order to investigate microcirculation in the two different ulcer areas, they were evaluated separately. Beside the two ulcer areas, a skin area was defined within a certain distance from the ulcer s border. A problem of this study was the variation of skin transparency, which is obviously more pronounced in ulcers due to necrotic material or detritus. The variation of skin transparency might influence the results of capillary microscopy more than those of laser Doppler flux measurements. To differentiate between a reduced capillary density in reality and that due to an impaired transparency, an additional capillary microscopy after the injection of a fluorescent dye (sodium fluorescin) was done in some patients. By means of this technique primarily non visible capillaries could possibly be discovered, but in our patients no relevant increase of capillary density was shown in the areas investigated. In addition, the number of capillaries among healthy individuals is strongly influenced by spatial differences from 30 to 50 in the dorsum of the foot to approximately 70 capillaries/mm 2 in toes [7,15]. This local variation has to be taken into account, when measurements of the laser Doppler flux are interpreted, too [16]. Another problem of the study was that investigations of patients were performed in supine position. Investigations were performed in this position, in order to enable capillary microscopy and laser Doppler scanning in the same test setup. But, if investigation of capillaries is performed in supine position, it might be argued, that in limb ischemia the number of visible capillaries might be lower than that in leg dependency. Some authors recommend therefore, that in ischemic skin, capillary microscopy should be performed in leg dependency, only [7]. Ubbink et al. [17] found a 4.5-fold increase of capillary density of the skin, in patients with limbthreatening ischemia, after leg dependency from supine position. However, the influence of the position of the legs on the capillary density within ulcers has not been examined so far. At least the relative differences in capillary density found here in the 3 regions of interest cannot be explained by the supine position. In addition, the main goal of the present study was to compare flux and capillary density of the different regions in a position, where laser Doppler imaging and capillary microscopy could be easily applied. Besides that, we did not determine biological zeros of flux, as an arterial arrest for 4.5 min (the maximum scanning time) in patients with ischemic ulcers was considered too painful and if three ROI of the same patient are compared, the relationship of the flux between the different ROI might not be substantially altered. From the results observed in this study, we conclude, that changes of perfusion of the deeper network of microcirculation as measured by a laser Doppler imager may coincide with changes of the number of visible surface capillaries within the ischemic ulcer, as blood of the skin capillaries is provided by subpapillary vessels. Acknowledgements We thank Professor G. Nilsson for the technical advice and Professor R. Horvat for the histologic examinations. References [1] Hoffmann U, Franzeck UK, Bollinger A. (Laser-Doppler technique in diseases of peripheral blood vessels) Laser-Doppler- Technik bei Krankheiten der peripheren Gefasse. Dtsch Med Wochenschr 1992;117: [2] Fagrell B. Advances in microcirculation network evaluation: an update. Int J Microcirc Clin Exp 1995;15(Suppl 1): [3] Fagrell B. Vital microscopy: a clinical method for evaluating the risk of skin necrosis in patients with occlusive arterial disease. Bibl Anat 1973;11: [4] Fagrell B. Vital capillary microscopy. A clinical method for studying changes of the nutritional skin capillaries in legs with arteriosclerosis obliterans. Scand J Clin Lab Invest Suppl 1973;133:2 50. [5] Fagrell B, Lundberg G. A simplified evaluation of vital capillary microscopy for predicting skin viability in patients with severe arterial insufficiency. Clin Physiol 1984;4: [6] Fagrell B. The skin microcirculation and the pathogenesis of ischaemic necrosis and gangrene. Scand J Clin Lab Invest 1977;37: [7] Bollinger A, Fagrell B. Clinical capillaroscopy. A guide to its use in clinical research and practice. Toronto: Hofgrefe and Huber, [8] Almond NE. Laser Doppler flowmetry: theory and practice. In: Belcaro G, Hoffmann U, Bollinger A, Nicolaides AN, editors. Laser Doppler. London: Med Orion, 1994: [9] Wardell K, Jakobsson A, Nilsson GE. Laser Doppler perfusion imaging by dynamic light scattering. IEEE Trans Biomed Eng 1993;40: [10] Gschwandtner ME, Koppensteiner R, Maca T, Minar E, Schneider B, Schnurer G, et al. Spontaneous laser doppler flux distribution in ischemic ulcers and the effect of prostanoids: a crossover study comparing the acute action of prostaglandin E1 and iloprost vs saline. Microvasc Res 1996;51: [11] Carter SA. Ankle and toe systolic pressures comparison of value and limitations in arterial occlusive disease. Int Angiol 1992;11: [12] Second European Consensus Document on chronic critical leg ischemia. Circulation 1991;84 (4 Suppl):IV1 26. [13] Staxrud LE, Jakobsson A, Kvernebo K, Salerud EG. Spatial and temporal evaluation of locally induced skin trauma recorded with laser Doppler techniques. Microvasc Res 1996;51:69 79.

8 232 M.E. Gschwandtner et al. / Atherosclerosis 142 (1999) [14] Staxrud LE, Kvernebo K, Salerud EG. Acute effects of local tissue trauma on skin perfusion evaluated with laser-doppler flowmetry. Microvasc Res 1991;42: [15] Konecny U, Ehringer H, Jung M, Koppensteiner R, Minar E, Stumpflen A, et al. Mapping the capillary density of hands and feet in healthy probands. Vasa Suppl 1987;20: [16] Tenland T, Salerud EG, Nilsson GE, Oberg PA. Spatial and temporal variations in human skin blood flow. Int J Microcirc Clin Exp 1983;2: [17] Ubbink DT, Jacobs MJ, Slaaf DW, Tangelder GJ, Reneman RS. Capillary recruitment and pain relief on leg dependency in patients with severe lower limb ischemia. Circulation 1992;85: