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Am J Physiol Heart Circ Physiol 288: H1224-H1232, 2005. First published October 28, 2004; doi:10.1152/ajpheart.00640.2004
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Evolution of a "falx lunatica" in demarcation of critically ischemic myocutaneous tissue

Yves Harder,1,2 Michaela Amon,1 Mirko Georgi,1 Andrej Banic,2 Dominique Erni,2 and Michael D. Menger1

1Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany; and 2Department of Plastic, Reconstructive and Aesthetic Surgery, Inselspital, University of Berne, Berne, Switzerland

Submitted 28 June 2004 ; accepted in final form 20 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Using intravital microscopy in a chronic in vivo mouse model, we studied the demarcation of myocutaneous flaps and evaluated microvascular determinants for tissue survival and necrosis. Chronic ischemia resulted in a transition zone, characterized by a red fringe and a distally adjacent white falx, which defined the demarcation by dividing the proximally normal from the distally necrotic tissue. Tissue survival in the red zone was determined by hyperemia, as indicated by recovery of the transiently reduced functional capillary density, and capillary remodeling, including dilation, hyperperfusion, and increased tortuosity. Angiogenesis and neovascularization were not observed over the 10-day observation period. The white rim distal to the red zone, appearing as "falx lunatica," showed a progressive decrease of functional capillary density similar to that of the necrotic distal area but without desiccation, and thus transparency, of the tissue. Development of the distinct zones of the critically ischemic tissue could be predicted by partial tissue oxygen tension (Pt) analysis by the time of flap elevation. The falx lunatica evolved at a Pt between 6.2 ± 1.3 and 3.8 ± 0.7 mmHg, whereas tissue necrosis developed at <3.8 ± 0.7 mmHg. Histological analysis within the falx lunatica revealed interstitial edema formation and muscle fiber nuclear rarefaction but an absence of necrosis. We have thus demonstrated that ischemia-induced necrosis does not demarcate sharply from normal tissue but develops beside a fringe of tissue with capillary remodeling an adjacent falx lunatica that survives despite nutritive capillary perfusion failure, probably by direct oxygen diffusion.

ischemia; myocutaneous flap; necrosis; microcirculation; angiogenesis; partial tissue oxygen tension; capillary remodeling; capillary dilation; capillary tortuosity

EXTENSIVE SOFT TISSUE DEFECTS after trauma and ablative surgery require reconstructive procedures for wound closure, including random pattern flaps and extended pedicled flaps with randomized areas (22, 26). To carry out safe surgery and to be successful with these flaps, the procedure has to guarantee an adequate supply of blood and oxygen to the tissue (10, 33, 38). However, inappropriate anatomical arrangement of the vasculature of the flap and interruption of blood supply during flap dissection may cause ischemia and necrosis, particularly in the most peripheral parts of the flap. This in turn remarkably increases the rate of partial flap loss and the morbidity of patients in plastic and reconstructive surgery (9, 33, 34).

To improve blood supply, tissue oxygenation, and, thus, survival of critically ischemic myocutaneous tissue, it is a prerequisite to understand the mechanisms that lead to ischemic flap complications in general and to define the processes that occur in the critical perfusion zone of the flap in particular. Studying focal ischemia in the brain, Astrup et al. (3) demonstrated that tissue that surrounds and thus demarcates an ischemic infarction is perfused at a level within the thresholds of functional impairment and morphological integrity. According to the half-shaded zone surrounding a complete solar eclipse, this part of the ischemic brain has been termed "penumbra." The critically perfused penumbral tissue is thought to be potentially salvageable if nutritive perfusion is adequately restored within a defined period of time (15).

In myocutaneous tissue, little is known of the mechanisms that determine the development of ischemic necrosis. In clinical practice it is thought that necrosis sharply demarcates from normal tissue; however, the demarcation zone itself and the factors that may provide survival from ischemia, such as angiogenesis and microvascular remodeling, have not been studied yet. The aim of the present study was therefore 1) to characterize the critically ischemic zone of demarcation in myocutaneous flaps and 2) to evaluate microvascular mechanisms that determine tissue survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experiments were performed according to the guiding principles for research involving animals and the German legislation on protection of animals. The experiments were approved by the local governmental animal care committee. A total of 22 mice (C57BL/6J, 12–24 wk, 24–26 g body wt; Charles River Laboratories, Sulzfeld, Germany) were included in the study. The animals were housed in single cages at a room temperature of 22–24°C and at a relative humidity of 60–65% with a 12:12-h light-dark cycle. The animals were allowed free access to drinking water and standard laboratory chow (Altromin, Lage, Germany).

Anesthesia. For surgery and repetitive intravital fluorescence microscopy, the animals were anesthetized by intraperitoneal injection of 90 mg/kg body wt ketamine hydrochloride (Ketavet; Parke Davis, Freiburg, Germany) and 25 mg/kg body wt xylazine hydrochloride (Rompun; Bayer, Leverkusen, Germany).

Preparation of myocutaneous flap. Surgery was performed by the first two authors throughout the series of experiments. The flap was prepared at the dorsum of the animal and was incorporated into a dorsal skinfold chamber, which was described previously in detail (20). In brief, after intraperitoneal anesthesia and removal of the fur, a laterally based myocutaneous flap with a width-to-length ratio of 15 x 11 mm, including the panniculus carnosus, was elevated perpendicularly to the spine, transsecting both the deep circumflex iliac artery (DCIA) and the lateral thoracic artery (LTA) (14). The flap and its neighboring skinfold tissue were then sutured into a skinfold chamber, which consisted of two symmetrical titanium frames (28). This was done in such a fashion that the observation window, incorporated in one of the frames, allowed direct view onto the muscle and subcutaneous tissue of the elevated flap. To avoid drying of tissue and influence of ambient air, the window was sealed with a cover glass. Chambers (total weight ~3 g) were well tolerated by the animals, which showed no changes in sleeping or feeding habits.

Preparation of dorsal skinfold chamber. In animals that served as controls, a dorsal skinfold chamber was mounted without flap elevation. Thus the tissue exposed within the observation window (also consisting of epidermis, subcutis, and striated muscle) was adequately perfused by both the DCIA and the LTA (20).

Intravital fluorescence microscopy. For in vivo microscopic analysis of the microcirculation, anesthetized mice were placed in a left lateral decubital position on a Plexiglas pad and received an intravenous injection of 0.05 ml of 5% fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight 150,000; Sigma Aldrich, Deisenhofen, Germany) via a tail vein. Subsequently, the mice were positioned under a Zeiss Axiotech microscope (Oberkocchen, Germany). The epi-illumination microscope setup included a 100-W mercury lamp and filter sets for blue (450–490 nm, excitation wavelength; >520 nm, emission wavelength), green (530–560 nm; >580 nm), and ultraviolet light (330–390 nm; >430 nm). Microscopic images were recorded using a charge-coupled device video camera (FK6990; Pieper, Schwerte, Germany) on video tape (Panasonic AG-7350-SVHS; Matsushita, Tokyo, Japan) for later off-line evaluation.

Analysis of microcirculation. The microscopy procedures for analysis of the microcirculation were performed at a constant room temperature of 23°C. Different objectives (x4, NA 0.16; x10, NA 0.30; and x20 long distance, NA 0.32) were used for recordings. At each observation time point, the tissue within the window of the chamber was scanned using the x4 objective to determine the surface of nonperfused and necrotic tissue, respectively. Furthermore, second- or third-order arterioles and accompanying collecting venules with easily identifiable branching patterns were selected in the proximal, middle, and distal parts of the flap. With the x10 and x20 objectives, video printouts of the above-described areas, including arteriolovenular bundles, were generated to indicate the exact localizations for repetitive measurements of red blood cell velocity, capillary diameter, volumetric blood flow, functional capillary density, and tortuosity of the vessels (39).

All parameters were analyzed off-line using a computer-assisted image analysis system (CapImage; Zeintl Software, Heidelberg, Germany) (17). The area of the flaps was determined planimetrically. The functional capillary density was defined as the length of red blood cell-perfused capillaries per observation field (expressed in cm/cm2). After selection of two individual branching points, tortuosity of capillaries was calculated from the ratio of the actual path length and the straight line, i.e., the shortest distance between the two branching points. Capillary diameters were measured perpendicularly to the vessel path. Capillary red blood cell velocity was analyzed using the line shift method (CapImage). Volumetric blood flow was calculated in arterioles, capillaries, and venules from red blood cell velocity (V) and vessel cross-sectional area ({pi}r2) according to the equation of Gross and Aroesty, i.e., Q = V{pi}r2, assuming a cylindrical vessel shape (12).

Analysis of skin oxygenation. Because the question of whether measurement of partial tissue oxygen tension (Pt) can predict subsequent tissue necrosis is of clinical interest, we measured Pt from the distal end to the base of the flap directly after elevation. Pt was analyzed using a flexible polyethylene microcatheter Clark-type PO2 device (LICOX system; GMS, Kiel-Mielkendorf, Germany) that was implanted between the skin and panniculus carnosus (n = 6). During measurement, the flap was covered with an oxygen-impermeable foil to prevent drying and exposure to ambient air. Online temperature compensation was performed by a temperature probe (type K thermocouple probe, LICOX system; GMS) that was positioned between the skin surface and the oxygen-impermeable foil. The probe was implanted in 2-mm steps from the distal end of the flap toward its base. After 45 min of equilibration, three different measurements were taken per probe position. Measurements in normal skin at the contralateral site served as controls.

Histological examination. Formalin-fixed, paraffin-embedded, full-thickness longitudinal segments of the flaps obtained at day 10 after surgery were cut (3.5-µm thickness) and stained with hematoxylin and eosin. The sections served for the examination of morphological changes within the epidermis, subcutaneous tissue, and panniculus carnosus. Within the panniculus carnosus, kariolysis was quantitatively assessed by counting the number of visible nuclei per square millimeter of surface. In parallel, edema formation was analyzed planimetrically from these histological sections by determining the area of the interstitial space, which is given as a percentage of the overall tissue area.

Experimental protocol. Eight animals were assigned to the experimental myocutaneous flap group. Repetitive microscopic observations as described above were performed 24 h as well as 3, 5, 7 and 10 days after surgery. An additional eight animals served as controls. At the end of the experiments, the animals were euthanized by injection of an overdose of the anesthetic. Another six animals were used to assess tissue oxygenation within the flap and the contralateral control site on the day of surgery.

Statistical analysis. All values are expressed as means ± SE. For comparison between individual time points, ANOVA followed by the appropriate post hoc test was performed, including correction of the alpha error according to Bonferroni probabilities. Comparison between groups was done using Student's t-test (SigmaStat; Jandel, San Rafael, CA). A value of P < 0.05 was taken to represent statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Macroscopic observation of the chamber's window over time demonstrated distinct microvascular deteriorations in the middle and distal areas of the flaps (Fig. 1). Most interestingly, we could observe a progressive demarcation of the flap in three distinct zones from distal to proximal. At day 1 after flap elevation, a transparent, nonperfused tissue area (transparent zone) within the most distal part of the flap became visible, indicating the progress of necrosis (Fig. 1A). At day 3, petechial bleedings developed within the middle area of the flap, appearing as a cloudy red zone (Fig. 1B). At day 7, the petechiae had resolved; however, a red fringe (red zone), attributed to microvascular hyperemia and remodeling, became apparent. Beside this, a white falx with rarefaction of microvessels (white zone) had developed, delineating the red zone of the middle area from the necrosis of the distal area of the flap (Fig. 1C). At day 10, demarcation of the three distinct zones adjacent to the normal tissue of the base of the flaps had fully developed (Fig. 1D) and remained unchanged until day 16 (data not shown). Length measurements from the base toward the distal end of the flap revealed that the unaffected normal tissue at the base extended from 0 to 6.1 mm, the red zone from 6.1 to 7.4 mm, the white zone from 7.4 to 9.0 mm, and the transparent zone, i.e., necrotic zone, >9.0 mm.



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  		Fig. 1. Observation windows revealing the flap tissue at days 1 (A), 3 (B), 7 (C), and 10 (D) after elevation. Note the progress of necrosis within the most distal area of the flap (A, asterisk), indicated by the increased transparency of the tissue. At day 3, petechial bleedings have developed within the middle part of the flap, appearing as a cloudy red zone (B, double arrows). At day 7, the petechiae are resolved, but, instead, a hyperemia-associated red fringe (red zone) has developed (C, double arrows). Beside this, a white falx with apparent rarefaction of microvessels (white zone) has appeared, delineating the red zone of the middle part from the necrosis of the distal part of the flap (C, arrow). At day 10, demarcation of the 3 distinct zones adjacent to the normal tissue of the flap's base has fully developed (D).

 
Intravital fluorescence microscopy. The corresponding in vivo microscopic imaging demonstrated the microvascular correlates to these functional and morphological changes. At day 1, the distal area of the flap was characterized by failure of perfusion of nutritive capillaries despite patent arterioles and venules (Fig. 2A). At days 3 and 5, a progressive loss of microperfusion was observed, including the capillaries of the middle part as well as the capillaries and the arteriolovenular bundles of the distal area of the flaps (Fig. 2B). At day 10, the transiently nonperfused capillaries within the red zone were found to be reperfused and dilated, whereas the capillaries within the distally adjacent white zone remained unperfused (Fig. 2C). Quantitative analysis of nonperfused tissue (white zone and transparent zone) revealed a total area of 43 ± 3% at day 10 after flap elevation. Interestingly, in none of the areas of the flaps could the development of new microvessels, including characteristic signs of angiogenesis such as capillary sprouting and bud formation, be observed over the 10-day observation period.



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  		Fig. 2. Intravital fluorescence microscopy of microvascular perfusion in the zone of transition from proximal (normal) toward distal (necrosis) at days 1 (A), 5 (B), and 10 (C) after flap elevation. Contrast enhancement was achieved with FITC-dextran 150,000 (magnification x25). The proximal area (normal) does not show any changes of microvascular perfusion over the 10-day observation period. In contrast, at day 1, the distal area of the flap was characterized by failure of perfusion of nutritive capillaries despite patent arterioles and venules (A, asterisk). At day 5, a progressive loss of microperfusion was observed, including the capillaries of the red zone (B, arrow) as well as the capillaries and the arteriolovenular bundles of the distal part (white zone and necrosis) of the flaps (B, double asterisk). At day 10, the transiently nonperfused capillaries within the red zone were reperfused and dilated (C, arrow), whereas the capillaries within the distally adjacent white and necrotic zone remained unperfused (C, double asterisk).

 
. Two-millimeter stepwise measurements of along the flap from the base toward its distal end showed a gradual decrease from 38.8 ± 3.2 to 1.5 ± 0.3 mmHg (P < 0.001; Fig. 3). The corresponding contralateral control site showed a of 41.1 ± 1.7 mmHg (not significantly different from the base of the flap). Correlation of those initial values at the time of flap elevation with the manifestation of the individual zones of demarcation at day 10 demonstrated that 1) the tissue of the base of the flap (proximal area) remained unaffected with a between 38.8 ± 3.2 and 10.3 ± 2.4 mmHg; 2) the red zone with dilated microvessels developed with a between 10.3 ± 2.4 and 6.2 ± 1.3 mmHg; 3) the almost avascular white zone developed with a between 6.2 ± 1.3 and 3.8 ± 0.7 mmHg; and 4) the transparent zone (distal area), which reflects necrosis, developed with a of <3.8 ± 0.7 mmHg (Fig. 3).



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  		Fig. 3. Measurements of partial tissue oxygen tension () directly after flap elevation show a gradual decrease along the flap from the base toward its distal end. The graph also displays manifestation of the individual zones at day 10, indicating that 1) the tissue at the base of the flap (proximal area, normal) remains unaffected with a PtO2 between 38.8 ± 3.2 and 10.3 ± 2.4 mmHg, 2) the vascularly dilated red zone develops with a PtO2 of <10.3 ± 2.4 mmHg, 3) the almost avascular white zone develops with a PtO2 of <6.2 ± 1.3 mmHg, and 4) the transparent zone, which reflects necrosis, develops with a PtO2 of <3.8 ± 0.7 mmHg. Values are means ± SE. *P < 0.05; **P < 0.001 vs. control and flap base.

 
Capillary perfusion. Within the proximal area of the flap, functional capillary density did not change over the entire 10-day observation period (Fig. 4, A and D). In contrast, the capillary density within the red zone was significantly (P < 0.05) reduced by ~35% at day 1 compared with that of the proximal area. From day 3 to day 7, capillary density further decreased (P < 0.05); however, it recovered at day 10 with values not significantly different from those of the proximal zone (Fig. 4, B and E). The capillary density of the white zone and the transparent zone was markedly reduced by ~80% at day 1 compared with that of the proximal area (P < 0.001) and did not recover during the further observation period but decreased until day 10 after flap elevation to almost zero and zero, respectively (Fig. 4, C and F).



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  		Fig. 4. A–C: intravital fluorescence microscopy of capillary perfusion at day 10 after flap elevation. Contrast enhancement was achieved with FITC-dextran 150,000 (magnification x100). Note the normal aspect of the capillaries at the flap's base (A), some rarefaction within the red zone (B), and dilated capillaries with an increase in tortuosity and sluggish blood flow at the transition between red zone and white zone (C). D–F: quantitative analysis of functional capillary density (FCD) indicates stable perfusion over the 10-day observation period in the proximal area of the flap (D), a transient decrease of perfusion until day 7 with recovery at day 10 in the red zone (E), and a progressive decrease of perfusion to almost zero without recovery in the white zone (F). G–I: capillary diameters of the perfused capillaries only slightly increase within the proximal area of the flap (G) but markedly increase within the red zone (H) and, in particular, the white zone (I) of the middle part of the flap. J–L: accordingly, tortuosity of the perfused capillaries also shows only a slight increase within the proximal area of the flap (J) but a marked increase within the red zone (K) and, in particular, the white zone (L). Values are means ± SE. *P < 0.05; **P < 0.001 vs. day 1 after flap elevation.

 
Analysis of capillary diameters and capillary tortuosity within the proximal area of the flaps showed only a slight increase over the 10-day observation period (Fig. 4, G and J). At day 1 after flap elevation, capillary diameters and tortuosity within the red and white zones did not differ from those in the proximal area (Fig. 4, H, I, K and L). However, from day 3 to day 10, these capillaries showed a marked dilation, presenting with a 2- and 3-fold increase in diameter (P < 0.001 and P < 0.05, respectively) in the red and white zones, respectively (Fig. 4, H and I). In parallel, the capillary tortuosity increased 5- and 14-fold (P < 0.05 and P < 0.001, respectively) until day 10 (Fig. 4, K and L).

At day 1 after flap elevation, capillary red blood cell velocity was significantly lower than that of controls (P < 0.001) in all parts of the flaps. Within the proximal and the middle areas (red zone and white zone), red blood cell velocity in those capillaries that still conducted flow recovered over time, whereas in the distal area no capillaries with perfusion could be detected during the further observation period (Table 1).


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  		Table 1. Red blood cell velocity of perfused capillaries in tissue after flap elevation

 
Analysis of individual volumetric blood flow in these capillaries showed reduced values compared with controls at day 1 after flap elevation, particularly within the red zone and white zone of the middle area of the flaps (P < 0.001) (Table 2). However, during the later time course, the still-perfused capillaries showed a hyperemic response with a 3- and 10-fold increase in volumetric blood flow (P < 0.05) in the red zone and white zone, respectively, which was mainly due to the marked dilatory response (Table 2). In the distal area of the flap, volumetric blood flow of still-perfused capillaries at day 1 was markedly decreased (P < 0.001), whereas no perfused capillaries could be detected during the later observation course (Table 2).


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  		Table 2. Individual volumetric blood flow of perfused capillaries in tissue after flap elevation

 
Tissue morphology. Within the proximal area of the flaps, all layers of the skin (epidermis, subcutaneous tissue, panniculus carnosus) appeared unaffected, as indicated by intact nuclei and lack of edema formation compared with controls without flaps (Fig. 5, A, E, and F). In contrast, analysis of the red zone showed interstitial edema formation within the panniculus carnosus (P < 0.001 vs. control) and marked dilation of microvessels with some congestion, as reflected by accumulation of red blood cells (Fig. 5, B and E). In addition, fibers of the panniculus carnosus showed some nuclear rarefaction, as indicated by a reduced number of nuclei per surface area (P < 0.05) (Fig. 5, B and F). Analysis of the white zone revealed edema formation to a lesser extent (P < 0.05) but a more pronounced nuclear rarefaction (P < 0.05) (Fig. 5, C, E, and F). Finally, the transparent zone was characterized by complete kariolysis within the panniculus carnosus (P < 0.001), associated with massive structural damage and shrinkage of the muscle fibers (Fig. 5, D–F).



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  		Fig. 5. A–D: longitudinal sections of flap tissue at day 10 after elevation stained with hematoxylin and eosin (magnification x160). In the proximal area of the flap, all layers of the skin (epidermis, subcutaneous tissue, and panniculus carnosus) appeared unaffected, as indicated by intact nuclei and lack of edema formation compared with controls without flaps (A, E, and F). Analysis of the red zone showed interstitial edema formation within the panniculus carnosus and marked dilation of microvessels with some congestion, as reflected by accumulation of red blood cells (B and E). In addition, fibers of the panniculus carnosus showed some nuclear rarefaction, as indicated by a reduced number of nuclei per surface area (B and F). Analysis of the white zone revealed edema formation to a lesser extent but more pronounced nuclear rarefaction (C, E, and F). Finally, the transparent zone was characterized by complete kariolysis within the panniculus carnosus, associated with massive structural damage and shrinkage of the muscle fibers (D–F). Open bars represent controls without flaps; filled bars represent flap elevation. Values in E and F are means ± SE. *P < 0.05; **P < 0.001 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows that the transition zone of critically ischemic myocutaneous flaps is characterized by a red fringe and a distally adjacent white falx that define the demarcation by dividing the normal from the necrotic tissue. The survival of the tissue in the red zone is determined by hyperemia and remodeling rather than by neovascularization. The tissue of the white zone lacks nutritive capillary perfusion, similarly to the necrotic area distally. However, the white zone does not show desiccation and, thus, transparency. Importantly, the development of the distinct zones of demarcation within the critically ischemic tissue at day 10 after flap elevation is already predictable at the time of flap elevation as show by analysis of partial tissue oxygen tension.

Although there is some information on demarcation of skin after electric and freeze-thaw injury (16, 31), no information is available on characteristics and mechanisms that determine survival and necrosis of tissue in critically ischemic conditions. However, this is of major interest, because demarcation of nonsurviving tissue due to ischemia is frequently observed in peripheral occlusive arterial disease as well as after complicated procedures in plastic, reconstructive, and vascular surgery (13, 18, 30, 32). In the present study, we have demonstrated that during evolution of ischemic necrosis in myocutaneous tissue, a critically perfused zone of demarcation, consisting of a red fringe and a white falx, develops between the adequately perfused proximal area and the nonperfused distal area condemned to necrosis.

Red zone development. Intravital microscopy showed that the development of the red zone was the consequence of a hyperemic capillary response. This response, however, cannot be interpreted as reactive hyperemia, which is known to occur immediately after short periods of ischemia (37, 40, 41). In the present study, the red zone did not develop immediately after flap elevation but only after a 7-day period. Accordingly, microscopy early after flap elevation showed a temporary decrease of functional capillary density and capillary red blood cell velocity, whereas capillary dilation was detected only at day 3 and hyperemic capillary perfusion only at days 7 and 10. Thus the capillary hyperemic response within the red zone has to be considered a delayed and chronic rather than an acute process, indicating remodeling that also has been described as a response mechanism to chronic hypoxia in the brain (7) and in the delay phenomenon of the skin (19).

Parallel to capillary dilation, we observed an increase of capillary tortuosity during development of the red zone. This also may be considered part of the process of remodeling and is probably driven by the chronic hypoxic conditions. It is worth noting that these dilated tortuous microvessels are autochthonous capillaries but not newly formed during the chronic ischemia. In peripheral occlusive arterial disease, which is also associated with chronic ischemia, collateral vessels also show a marked tortuous path (35). However, these vessels develop from arteriogenesis rather than angiogenesis, which is not driven by hypoxia (36), and present with an arteriolar rather than a capillary structure. During aging, remodeling of the microvasculature, as a consequence of progressive reduction of tissue oxygenation, also involves an increase in microvascular tortuosity. However, this process of remodeling is confined to the venular segments of the microvasculature (39). In fact, the increase in capillary tortuosity observed in the present study within the red fringe of the demarcation zone is most comparable with the increased tortuosity of cerebral capillaries induced by chronic hypobaric hypoxia (7). This type of remodeling in the brain is thought to significantly increase the O2 conductance to the neural tissue (7). In fact, this mechanism also may be responsible for the survival of the critically endangered myocutaneous tissue observed in the present study within the red zone of the flaps.

White zone development. Within the critically perfused transitional area another distinct zone developed distally to the red zone that appeared as a white falx, best described as "falx lunatica." Unlike the delayed vascular remodeling within the red zone, the white zone was characterized by a rarefaction of nutritive microvessels, as shown by a significant reduction of capillary density. This was apparent already at day 1 and progressed further to almost complete perfusion failure after the 10-day observation period. The lack of capillary perfusion may simply be the consequence of the failing arterial inflow to the distal area of the flap. Although perfusion failure within the falx lunatica was similarly pronounced compared with that of the distally adjacent transparent (necrotic) zone, these two zones showed marked morphological differences. The necrotic zone was characterized by thinning and translucency of the tissue due to cell disintegration and desiccation, representing irreversible injury. In contrast, the falx lunatica showed only minor cellular damage with an almost preserved tissue consistency and some interstitial edema, similar to what was found in the tissue of the red zone.

The development of such a zone in critically ischemic skin has not yet been described, but similarities to the ischemic penumbra in the brain are striking. Astrup and coworkers (4) demonstrated an ischemic cortical tissue that surrounded the irreversibly damaged core in focal cerebral ischemia. This cortical tissue had lost its function, yet it maintained most of its membrane integrity, suggesting functional failure, but viability (4). Characteristically, this functionally severely impaired tissue, evolving within a few hours after onset of stroke (11, 24), may escape ischemic necrosis if promptly reperfused; otherwise, it will be progressively recruited into the core of the necrotic zone until maximum infarction is achieved (3, 5, 21). In contrast, the falx lunatica, analyzed in the present study, develops during a 7-day period within the transition zone of the critically perfused myocutaneous tissue and then persists without treatment for at least 16 days after onset of the chronic ischemic conditions.

Thus the falx lunatica may represent a tissue with "vita minima." These conditions theoretically may be guaranteed by the few residual dilated capillaries that presented with perfusion. However, although these capillaries carried an up to 9-fold higher individual blood flow and their O2 conductance may be increased because of their increased tortuosity, the reduction of the functional capillary density to <5% of that of control tissue indicates that they do not serve for an adequate nutritional tissue supply but may function only as "through-fare" channels, as described for arteriolovenular shunts (29). More likely, the vita minima of the tissue of the falx lunatica is guaranteed by oxygen diffusion and interstitial space fluid equilibration, which may be capable of maintaining part of the cells viable and part of the tissue structure intact. With this view, the limited distance of oxygen diffusion may determine the size of the falx lunatica.

Angiogenesis. Although we observed remodeling of capillary diameter and length within the critically ischemic red and white zones, as also observed in hypoxic brain tissue (7), we could not demonstrate angiogenesis, i.e., capillary sprouting, vascular bud formation, and revascularization. In the brain, the formation of new capillaries is thought to be an important mechanism to restore the O2 deficit in chronic hypoxia (7). In parallel, chronic hypoxia in peripheral occlusive arterial disease also has been shown to induce angiogenesis and neovascularization (8, 23). In critically ischemic skin, the role of angiogenesis is still controversially discussed (2, 42). Mild hypoxia as induced by the delay conditioning procedure may promote angiogenesis and new blood vessel formation (1, 6). However, the elevation of myocutaneous flaps, as done in the present study, may have induced a massive drop in below critical thresholds rather than mild hypoxia. Although hypoxia is thought to be an important cellular mechanism to promote vascular endothelial growth factor-mediated angiogenesis (25), a most severe hypoxic condition as well as complete anoxia may not be capable of stimulating a neovascularization response due to paralysis of cellular metabolism. Thus myocutaneous values of >10 mmHg may not be sufficiently low to stimulate new vessel formation, whereas critical hypoxia with values of 5 mmHg, such as those measured in the falx lunatica, may not be able to induce an appropriate growth response.

. In clinical practice it is still a matter of discussion whether the at the time of flap elevation can predict the area of flap necrosis (27, 38). In the present study, we demonstrated that an initial between 6 and 10 mmHg results in capillary remodeling and recovery of functional capillary density, which reliably can be associated with tissue survival. A skin of 4–6 mmHg may provide conditions for a vita minima, whereas a <4 mmHg inevitably results in manifestation of tissue necrosis.

In conclusion, we have demonstrated for the first time that critical ischemia-induced necrosis in myocutaneous flaps does not demarcate sharply from normal tissue. Between the adequately perfused tissue and the nonperfused necrotic tissue, a fringe of tissue with capillary remodeling develops a distally adjacent falx lunatica that lacks nutritive capillary perfusion and survives solely by oxygen diffusion.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
Y. Harder is the recipient of a fellowship from the Swiss National Science Foundation (SNF-no. PBBSB-102601), the Freiwillige Akademische Gesellschaft, and the Margarete und Walter Lichtenstein Stiftung, Medical Department, Basel, Switzerland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Harder, Institute for Clinical and Experimental Surgery, Univ. of Saarland, D-66421 Homburg/Saar, Germany (E-mail: yvesharder{at}bluewin.ch)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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