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1Institute for Clinical and Experimental Surgery and 2Department of Anesthesiology and Intensive Care Medicine, University of Saarland, 66421 Homburg/Saar, Germany
Submitted 1 December 2003 ; accepted in final form 15 June 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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microcirculation; heat shock protein-32; tissue necrosis; skin flap; mouse ear; carbon monoxide; vitamin E analog
Although these studies have shown improvement of flap survival independent of the nature of the conditioning procedure, pharmacological interventions may vary in effectiveness to enhance flap survival and may in general not achieve the degree of tissue protection, which is observed after surgical conditioning procedures (22, 31). Thus surgical ischemic conditioning has been suggested the predominant experimental and clinical method to improve flap survival (6, 32).
Surgical ischemic conditioning of the tissue may be done by the classic ischemic preconditioning or by inducing the delay phenomenon. Ischemic preconditioning consists of a short but complete ischemia, which is performed intraoperatively for a few minutes by clipping the main feeding vessels just before final flap creation (1). In contrast, the delay phenomenon is induced by moderate CI through ligation of individual feeding vessels several days before flap creation (39). Despite the fact that the latter procedure has been used for many years in clinical practice, the mechanisms of the delay phenomenon in protecting tissue against necrosis are still poorly understood. Previous studies (3, 12, 46) have indicated that the delay acts via maintenance of an adequate microcirculation and adaptive metabolic changes at a cellular level, resulting in improved tolerance against a subsequent ischemic insult. However, the molecular mechanisms of protection of tissue by the delay phenomenon have not been elucidated yet.
Heme oxygenase (HO)-1 may play an important role in the delay-mediated protection of tissue. This microsomal enzyme catalyzes the initial and rate limiting reaction in heme catabolism, i.e., the oxidative cleavage of the 
-meso-carbon bridge of 
-type heme molecules to yield equimolar quantities of biliverdin-IXa, carbon monoxide (CO), and iron (47). Biliverdin-IXa reduced to bilirubin acts as a potent antioxidant, which protects cells against oxidative stress (26, 42). CO, a further product of the pathway, acts like nitric oxide, on soluble guanylyl cyclase, increasing cGMP, a second messenger involved in the regulation of multiple cell functions, and thereby contributing to the regulation of vascular tone (16, 29). Heme oxygenase exists in three isoforms, i.e., HO-1, HO-2, and HO-3 (8). Among the three isoforms, HO-1 can be induced substantially and has been identified as the major 32-kDa heat shock (stress) protein HSP-32 (25). Like other stress proteins, HO-1 can be induced by a variety of apparently disparate stimuli, most of which are linked by their ability to provoke oxidative stress (8).
As the result of the above-mentioned properties of the enzyme system, it is conceivable to hypothesize that the protective effect of the delay phenomenon is mediated by induction of HO-1. Therefore, we investigated whether 1) conditioning of a skin flap by CI induces HO-1 expression, 2) the activity of this enzyme improves flap survival, and 3) vasoactive or anti-oxidative actions of the enzyme system mediate delay-associated tissue protection.
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MATERIALS AND METHODS |
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The mouse ear consists of a single layer of cartilage sandwiched between two full dermal layers of skin (overall thickness 300 µm) and is completely hairless, which makes it ideal for intravital microscopic studies. Blood supply is provided by three vascular bundles, entering the ear at its base and forming an interconnecting capillary network at the periphery (5).
The animals were housed in individual cages and had access to standard laboratory chow (Altromin) and water ad libitum. For induction of the delay phenomenon and flap creation as well as for laser Doppler flowmetry and microscopic analysis, the mice were anesthetized with an intraperitoneally administered mixture of ketamine and xylazine (90 and 25 mg/kg body wt).
Induction of the delay phenomenon and creation of the skin flaps. After anesthesia, the ear to be investigated was gently extended over a microscopic slide embedded into a Plexiglas pad. The delay phenomenon was provoked by local CI induced by transsection of the central feeding vessel of the ear 7 days before flap creation (3).
To create an arterial pattern skin flap, the ear was incised through four-fifths of its base, rendering it dependent solely on the anterior vessel for its nourishment. The creation of the skin flap is associated with nutritive perfusion failure, and, within a 5-day observation period, development of tissue necrosis encompassing 
40% of the overall area of the ear (Fig. 1) (4).
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Intravital fluorescence microscopy.
For the in vivo microscopic procedure, the ears were flattened on the Plexiglas pad. In the proximal, central, and distal part of the flap, microcirculatory parameters and tissue necrosis were analyzed by intravital fluorescence microscopy with the use of a Leitz Orthoplan microscope. Microscopy was performed at a constant room temperature of 23°C. Contrast enhancement for visualization of the microcirculation was achieved by injection of 0.1 ml 5% FITC-dextran 150,000 (Sigma Aldrich; Deisenhofen, Germany) into a tail vein. The dye binds neither to endothelial cells nor to individual blood cells and does not extravasate under physiological conditions. Because of its high molecular weight, it remains within the intravascular space for an 4-h period and is then cleared from the circulation by the liver and the kidney. All measurements were done after the ears were embedded on a Plexiglas pad and fixed with an oxygen-impermeable foil.
The microscopic epi-illumination setup included a 100-W mercury lamp and a Ploemopak illuminator equipped with an I2 blue filter (450–490 nm excitation/>515 nm emission wave lengths). The movement of the microscope platform in x/y-directions was achieved manually, which allowed measurements in five positions in each part of the ear, comparable to the laser Doppler fluxmetry analysis. Microscopic images were recorded by a charge-coupled device video camera (CF8/1 FMC, Kappa; Gleichen, Germany) and were stored on videotape for subsequent off-line evaluation.
Analysis of microcirculation and tissue necrosis.
All microcirculatory parameters, i.e., capillary diameter, functional capillary density, red blood cell velocity (RBV), volumetric blood flow (VBF), and tissue necrosis were quantified off-line by analysis of the videotaped images using a computer-assisted image analysis system (CapImage; Zeintl Software; Heidelberg, Germany). RBV was assessed by the line shift method (2). Necrosis was quantified by determining the ratio of tissue area with nonperfused capillaries divided by the total tissue area of the ear (4). Capillary diameters were measured perpendicularly to the vessel path (in µm) only in red blood cell-perfused capillaries. Functional capillary density was defined as the length of all red blood cell-perfused capillaries per area of skin surface (cm/cm2) (28). Individual VBF was calculated in capillaries from RBV and vessel cross-sectional area (
·r2) according to the equation VBF = RBV··r2, assuming cylindrical vessel geometry. Both RBV and VBF are presented as a percentage of the baseline to demonstrate the changes during the observation time.
Western blot analysis of HO-1. Pooled protein (100 µg/lane) from ear tissue homogenates (4 ears/group) were subjected to SDS-PAGE under denaturing conditions and transferred to polyvinylidene difluoride membranes. The blot was probed with polyclonal rabbit anti-rat HO-1 antibody (dilution: 1:1,000; StressGen Biotechnologies; Sidney, BC, Canada) after blocking of unspecific binding sites with a mixture of 5% nonfat dry milk in 20 mM tris(hydroxymethyl)aminomethane, pH 7.5, 0.5 M NaCl, and 0.1% Tween 20. A horseradish peroxidase-linked donkey anti-rabbit secondary antibody (dilution: 1:10,000) was used to detect protein-antibody complexes with a chemiluminscent reaction (enhanced chemiluminescence Western blot analysis system; Amersham Buchler; Braunschweig, Germany) according to the manufacturer's instructions.
Immunohistochemistry of HO-1. Cell-type-specific expression and spatial pattern of HO-1 induction was studied in paraffin-embedded dewaxed sections essentially as described previously (7). Briefly, after incubation of sections in 3% H2O2-methanol to block endogenous peroxidase activity, slides were incubated with normal goat serum, followed by the primary HO-1 antibody used for Western blot analysis (dilution 1:200) at 37°C for 1 h. A biotinylated goat anti-rabbit antibody was used as secondary antibody for streptavidin-biotin complex peroxidase staining. 3,3'-Diaminobenzidine and 3% NiCl2 were used as chromogens, and slides were counterstained with hematoxylin.
Chemicals. Tin-protoporphyrin-IX (SnPP-IX, Porphyrin Products; Logan, UT) was dissolved by mixing SnPP-IX with sodium bicarbonate 8.4% and phosphate-buffered saline to achieve a final concentration of 5 µmol/ml. The solution was stored at a maximum temperature of 8°C in a light-protected tube until usage for not >1 h. The water-soluble vitamin E analog Trolox (Sigma Aldrich; Steinheim, Germany) was dissolved by mixing 97% 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carbonsacid and 0.9% saline. All solutions were freshly prepared at the day of the experiment according to the directions for use given by the manufacturer.
Experimental protocol. Tissue necrosis and microvascular perfusion in the proximal, central, and distal part of the flaps were determined before conditioning, immediately after skin flap creation, and, thereafter, daily over a 5-day period. After baseline measurements, the induction of CI or flap creation was performed as described before. After the induction of CI, animals of the first group were not further pretreated (CI, n = 8). To study the role of HO-1 in this model, animals of the second group received the false substrate SnPP-IX (50 µmol/kg body wt ip; CI+SnPP-IX; n = 8) to block HO-1 activity before the induction of CI and directly before flap creation. To test whether the antioxidative action of HO-1 is responsible for tissue protection, animals of the third group received beside SnPP-IX the vitamin E analog trolox (6 mg/kg body wt iv, every 12 h; CI+SnPP-IX+Trolox, n = 8) at the time of flap creation. Animals with flap creation but without any treatment served as controls (Control, n = 8). The substance-specific effects of SnPP-IX and trolox on microcirculation and flap necrosis were additionally assessed in animals without chronic ischemic conditioning (Control+SnPP-IX and Control+Trolox, both n = 8).
Statistical analysis. Data are given as means ± SE. After the assumption of normality was proven, comparison between the experimental groups was performed by one-way ANOVA, followed by the appropriate post hoc comparison test. P < 0.05 was considered to indicate significant differences.
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RESULTS |
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In the proximal part of the ear, flap creation resulted in a minor capillary dilation (Fig. 5A), which was associated with a slight alteration of microvascular perfusion, including red blood cell flux (Fig. 6A), as determined by laser Doppler flowmetry, and functional capillary density (Fig. 7A), capillary RBV (Fig. 8A), and capillary VBF (Fig. 9A), as analyzed by intravital microscopy. CI-conditioned animals showed also minor capillary dilation (Fig. 5A) and a decrease of functional capillary density (Fig. 7A); however, these animals maintained overall red blood cell flux (Fig. 6A) and capillary RBV (Fig. 8A), resulting in a higher individual capillary VBF when compared with that of nonconditioned controls (Fig. 9A). Blockade of HO-1 by SnPP-IX completely abrogated these beneficial effects of delay, and additional administration of Trolox did not affect the SnPP-IX-induced abrogation of microcirculatory protection (Figs. 6A, 8A, and 9A).
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In the distal part of the ear, flap creation induced marked capillary dilation (Figs. 5B and 10), which was associated with a massive alteration of microvascular perfusion to <10% of baseline, including red blood cell flux (Fig. 6B), functional capillary density (Fig. 7B), capillary RBV (Fig. 8B), and capillary VBF (Fig. 9B). Most strikingly, CI (delay) further pronounced capillary dilation (Figs. 5B and 10D) and was effective to significantly attenuate the decrease of red blood cell flux (Fig. 6B), functional capillary density (Fig. 7B), and capillary RBV (Fig. 8B). This resulted in complete maintenance of capillary VBF (Fig. 9B). Blockade of HO-1 by SnPP-IX abrogated capillary dilation (Figs. 5B and 10F) and completely blunted the CI-associated attenuation of microvascular perfusion failure, including red blood cell flux (Fig. 6B), functional capillary density (Fig. 7B), capillary RBV (Fig. 8B), and, thus capillary VBF (Fig. 9B). Trolox did not restore the SnPP-IX-mediated loss of protection from microcirculatory dysfunction (Figs. 5B, 6B, 7B, 8B, 9B, and 10H).
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DISCUSSION |
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CI strongly induces gene expression of HO-1. Since 1968, when the fist heme oxygenase (HO) system was detected (47), strong efforts have been made to characterize the function of this enzyme. HO represents the most efficient system to catalyze the oxidative cleavage of heme to equimolar amounts of biliverdin, CO, and iron (8, 47). Meanwhile, three isoforms of HO have been identified. The noninducible HO-2, constitutively expressed in most tissues, including the brain, testis, and liver (7, 26), seems to be of particular importance for cell integrity and cell function. The recently discovered, barely known isoform HO-3, which is expressed at a very low level, seems to be responsible for intracellular binding and transport of iron (15, 27). The isoform HO-1 is an enzyme of particular interest because of its identification as HSP-32 (25, 38). Like all other stress proteins, HO-1 is inducible by a variety of stress factors, e.g., heme (39), heat (38), hyperoxia but also hypoxia (36), and has been shown to be expressed in all tissues analyzed (26).
The present study demonstrates the induction of HO-1 in skin tissue by chronic hypoxia, probably triggered by the hypoxia promoter (13) on the 5' region of the HO-1 gene. Nonetheless, the generation of oxidative stress rather than the availability of distinct promotors seems to be the common trigger for HO-1 gene expression after exposure to potential inducers (8, 36). Thus production of oxygen radicals leads to an activation of oxygen-dependent transcription factors, such as activator protein-1 and NF-
B. Both are predominantly involved in the regulation of HO-1 gene transcription (8), supporting the widespread inducibility of this enzyme. This pathway may also be responsible for the induction of HO-1 expression by CI, which is thought to be the inducer of the delay phenomenon. Chronic hypoxia generates oxidative stress (14), which mediates HO-1 gene expression by oxygen-dependent transcription factors (8, 36). This view is supported by the present observation that flap creation without chronic ischemic conditioning also induced HO-1 expression over time, indicating that this procedure also resulted in tissue hypoxia, which activated HO-1 de novo expression.
HO-1 improves skin flap viability. Flap transfer is a widely used technique in plastic and reconstructive surgery. Although strong efforts have been made to understand flap pathophysiology (23) to develop novel strategies for improvement of flap viability, there is no distinct approach available to prevent flap necrosis. One of the most effective procedures to ameliorate flap viability is the induction of the delay phenomenon (31, 32). In fact, of the numerous types of conditioning procedures tested during the past two decades, only ischemic conditioning produced convincing results (24). Some studies (19, 45) have shown that ischemic pretreatment of critically perfused flaps augments blood supply, and, by this, advances tissue viability. A variety of theories have been proposed as the mechanisms for the augmented blood supply, including closure of shunts (34), increase of vascularization (6), and improvement of capillary perfusion (32). However, these microcirculatory changes have not been proven in vivo, and the molecular mechanisms of protection are not understood yet.
The results of the present study now demonstrate delay-induced maintenance of adequate nutritive capillary perfusion in vivo and indicate that the protective effect of the delay phenomenon is mediated by the action of HO-1, which catalyzes the cleavage of heme to CO, biliverdin, and free iron. CO and its action on capillary pericytes may play a major role in this delay-associated protection. It is well known that capillaries are associated abluminally with pericytes (21), which protrude primary cytoplasmic processes running along the capillary axis. From these primary processes, lateral processes arise, which completely encircle the capillaries and form tight connections to the endothelial cells through multiple contact points (10, 17, 21, 37, 40). These pericytes contain muscle cytoskeletal proteins, in particular, -smooth muscle actin (41), which are thought to regulate microvascular blood flow (21). In liver tissue, in vivo studies have shown that Ito cells, which are the sinusoid (liver capillary)-associated pericytes (44), are responsible for CO-mediated blood flow regulation in sinusoids, because blockade of CO resulted in sinusoidal constriction at the site of the Ito cells (43). This view was additionally confirmed in that the addition of CO, which is known to increase intracellular cGMP levels (29, 33), or the direct application of a cGMP analog resulted in attenuation of sinusoidal constriction (43). On the basis of this knowledge, increased CO levels, generated by elevated HO-1 activity, may explain the increase in capillary diameter, which ameliorates the blood supply of stress-exposed tissue, as indicated by increased red blood cell flux and VBF. In addition, there is evidence that HO-1 positively influences blood rheology (11), which could result in higher RBV because of unhindered flow. By this, tissue viability increases because of improved nutritional supply and better elimination of toxic residues of oxidative stress.
Biliverdin is subject to further degradation to bilirubin by the cytosolic enzyme biliverdin reductase (8, 26). It acts as an antioxidant and is capable to scavenge free oxygen radicals that are thought to be primarily responsible for the tissue injury (42). The additional application of Trolox, which mimics the antioxidative action of bilirubin, transiently delayed the development of necrosis, however, without improving the flap-associated microcirculatory disorders. This may be because bilirubin, produced by HO-1, inhibits cell damage by scavenging oxygen radicals subsequently after the stress event, thus improving tissue viability. Iron, the last product of heme cleveage, acts as a pro-oxidant like other transition metals and catalyzes the formation of reactive ·OH by the Fenton or Haber-Weiss reaction. These radicals are responsible for activation of cell death programs (8), which aggravate stress-induced cell demage. It seems to be of particular importance to eliminate free iron from the stressed tissue to maintain cellular integrity after the stress event. To enable this process, an additional expression of ferritin, the iron-binding protein, is induced simultaneously by HO-1 (8, 18).
The results of the present study show that chronic ischemic conditioning induces HO-1, augments blood flow, and, thus improves flap viability in the distal part of the flap, which is exposed to most critical hypoxia and probably highest levels of oxygen radicals. Expression of different heat shock proteins is a general response of cells to various types of stress (30) to prevent cell destruction. Although several of these stress proteins may be involved in providing tolerance against injury, HSP-70 has been thought to be most important for tissue protection under critical ischemic conditions (20). The present study, however, now indicates a major role of HO-1 for delay-induced improvement of flap survival, probably by reducing microcirculatory failure within the critically endangered tissue portions. This conclusion is based on the fact that inhibition of HO-1 by SnPP-IX almost completely blunted both delay-induced preservation of microcirculation and improvement of flap survival.
Delay-associated tissue protection is obtained by HO-1-mediated attenuation of microcirculatory dysfunction. The enhanced HO-1 activity may provide tissue protection by CO-mediated vasodilation or biliverdin-associated anti-oxidative actions (35). To elucidate the role of a solely anti-oxidative treatment, the vitamin E-analog Trolox was given after excluding the action of both CO and biliverdin by blockade of HO-1 with SnPP-IX. Although Trolox may not exactly mimic the action of biliverdin, its effects allow us to evaluate the role of reactive oxygen species in mediating flap microcirculatory dysfunction. As demonstrated in the present study, anti-oxidative Trolox treatment after selective blockade of HO-1 reduced necrosis only until day 3 after flap creation and did not affect the manifestation of microcirculatory disorders. This result indicates that oxidative stress may play some role in the development of initial flap necrosis, and that anti-oxidative therapy is capable to counteract this early oxidative stress-induced cell damage. However, it also demonstrates that the initial oxidative stress does not determine the final amount of flap necrosis, and that the overall protection from flap necrosis by chronic ischemic conditioning (delay) is mediated by HO-1 not through its anti-oxidative but rather through its vasodilatory action, preventing microvascular perfusion failure.
In summary, we conclude that the induction of HO-1 is an important mechanism by which the delay phenomenon provides protection to critically endangered tissue, and that the improvement of flap survival is mainly due to the efficacy of HO-1 to adequately maintain nutritive capillary perfusion.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-induced microcirculatory dysfunction and apoptotic cell death. FASEB J 17: 175–185, 2003.This article has been cited by other articles:
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  Y. Harder, M. Amon, M. Georgi, A. Banic, D. Erni, and M. D. Menger Evolution of a "falx lunatica" in demarcation of critically ischemic myocutaneous tissue Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1224 - H1232. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. CI+SnPP-IX+Trolox.
), after pretreatment with CI (delay; 
), and additional treatment with SnPP-IX (CI+SnPP-IX; 
) and Trolox (CI+SnPP-IX+Trolox; 
). Note the marked capillary dilation in controls and delayed flaps, which is less pronounced after inhibition of HO-1 by SnPP-IX. Values are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. CI+SnPP-IX; 
