Michaela Amon

Heme oxygenase and nitric oxide synthase mediate cooling-associated protection against TNF-α-induced microcirculatory dysfunction and apoptotic cell death

Local cooling protects against TNF‐α‐induced injury by attenuating inflammation‐associated microcirculatory dysfunction and leukocytic response. Mechanisms of protection, however, are not fully understood. We studied whether the metabolites of the HO and NOS pathway, exerting potent vasodilatory, antioxidant, and anti‐apoptotic properties, are involved in tissue cryoprotection. In animals pretreated with L‐NAME or SnPP‐IX, cooling‐associated abrogation of TNF‐α‐induced microcirculatory dysfunction was abolished. Combined L‐NAME/SnPP‐IX pretreatment did not cause greater blunting than seen when each mediator system was inhibited separately. In SnPP‐IX‐ but not L‐NAME‐pretreated animals, transient hypothermia failed to reduce TNF‐α‐mediated leukocyte adherence. Vice versa, treatment of TNF‐α‐exposed animals with either the NO donor l‐arginine or the HO‐1 inductor hemin mimicked cooling‐associated tissue protection except for failure of l‐arginine to abrogate the inflammatory leukocyte response. The efficiency of cooling to inhibit TNF‐α‐induced apoptotic cell death was blunted in SnPP‐IX‐, L‐NAME‐, and SnPP‐IX/L‐NAME‐pretreated animals. Coadministration of Trolox in SnPP‐IX‐treated animals partly attenuated leukocyte adherence and cell apoptosis, implying that the HO pathway metabolite biliverdin contributes to the salutary effects of cooling. Thus, our study provides evidence that metabolites of the HO and the NOS pathway mediate the cooling‐associated protection of inflamed tissue. Biliverdin rather than CO and NO mediates the anti‐inflammatory action, whereas a coordinated function of the gaseous monoxides prevents microcirculatory dysfunction and apoptotic cell death.—Amon, M., Menger, M. D., Vollmar, B. Heme oxygenase and nitric oxide synthase mediate cooling‐associated protection against TNF‐α‐induced microcirculatory dysfunction and apoptotic cell death. FASEB J. 17, 175–185 (2003)

Heat Shock Preconditioning Reduces Ischemic Tissue Necrosis by Heat Shock Protein (hsp)-32-mediated Improvement of the Microcirculation Rather Than Induction of Ischemic Tolerance

Introduction:Supraphysiologic stress induces a heat shock response, which may exert protection against ischemic necrosis. Herein we analyzed in vivo whether the induction of heat shock protein (HSP) 32 improves survival of chronically ischemic myocutaneous tissue, and whether this is based on amelioration of microvascular perfusion or induction of ischemic tolerance. Methods:The dorsal skin of mice was subjected to local heat preconditioning (n = 8) 24 hours before surgery. In additional heat-preconditioned animals (n = 8), HSP-32 was inhibited by tin-protoporphyrin-IX. Unconditioned animals served as controls (n = 8). A random-pattern myocutaneous flap was elevated in the back of the animals and fixed into a dorsal skinfold chamber. The microcirculation, edema formation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day period using intravital fluorescence microscopy. Results:HSP-32 protein expression was observed only in heat-preconditioned but not in unconditioned flaps. Heat preconditioning induced arteriolar dilation, which was associated with a significant improvement of both arteriolar blood flow and capillary perfusion in the distal part of the flap. Further, heat shock reduced interstitial edema formation, attenuated apoptotic cell death, and almost completely abrogated the development of flap necrosis (4% ± 1% versus controls: 53% ± 5%; P[r] < 0.001). Most strikingly, inhibition of HSP-32 by tin-protoporphyrin-IX completely blunted the preconditioning-induced improvement of microcirculation and resulted in manifestation of 72% ± 4% necrosis. Conclusion:Local heat preconditioning of myocutaneous tissue markedly increases flap survival by maintaining adequate nutritive perfusion rather than inducing ischemic tolerance. The protection is caused by the increased arteriolar blood flow due to significant arteriolar dilation, which is mediated through the carbon monoxide-associated vasoactive properties of HSP-32.

Erythropoietin protects critically perfused flap tissue

Objective:The objective of this study was to analyze whether erythropoietin (EPO) protects from necrosis of critically perfused musculocutaneous tissue and the mechanisms by which this protection is achieved. Background:EPO is the regulator of erythropoiesis and is used to treat patients with anemia of different causes. Recent studies suggest that EPO has also other tissue-protective effects, irrespective of its erythropoietic properties. Material and Methods:C57BL/6-mice were treated with 3 doses of EPO at 500 IU/kg intraperitoneally. EPO was given either before (preconditioning, n = 7), before and after (overlapping treatment, n = 7), or after (treatment, n = 7) surgery. Animals receiving only saline served as controls (CON). Acute persistent ischemia was induced by elevating a randomly perfused flap in the back of the animals. This critically perfused tissue demonstrates an initial microvascular failure of ∼40%, resulting in ∼50% tissue necrosis if kept untreated. Repetitive fluorescence microscopy was performed over 10 days, assessing angiogenesis, functional capillary density, inflammatory leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis. Additional molecular tissue analyses included the determination of inducible nitric oxide synthase, erythropoietin receptor (EPO-R), and vascular endothelial growth factor (VEGF). Results:EPO preconditioning did not affect hematocrit and EPO-R expression, but increased inducible nitric oxide synthase in the critically perfused tissue. This correlated with a significant arteriolar dilation, which resulted in a maintained functional capillary density (CON: 0 ± 0 cm/cm2; preconditioning: 37 ± 21 cm/cm2; overlapping treatment: 72 ± 26 cm/cm2; P < 0.05). EPO pretreatment further significantly reduced microvascular leukocyte adhesion and apoptotic cell death. Moreover, EPO pretreatment induced an early VEGF upregulation, which resulted in new capillary network formation (CON: 0 ± 0 cm/cm2; preconditioning: 40 ± 3 cm/cm2; overlapping treatment: 33 ± 3 cm/cm2; P < 0.05). Accordingly, EPO pretreatment significantly reduced tissue necrosis (CON: 48% ± 2%; preconditioning: 26% ± 3%; overlapping treatment: 20% ± 3%; P < 0.05). Of interest, EPO treatment was only able to alleviate ischemia-induced inflammation but could not improve microvascular perfusion and tissue survival. Conclusions:EPO pretreatment improves survival of critically perfused tissue by nitric oxide -mediated arteriolar dilation, protection of capillary perfusion, and VEGF-initiated new blood vessel formation.

Experimental models to study microcirculatory dysfunction in muscle ischemia–reperfusion and osteomyocutaneous flap transfer

BackgroundDuring the past decade, experimental studies have provided convincing evidence that microcirculatory dysfunction plays a pivotal role in the manifestation of tissue injury in ischemia–reperfusion and osteomyocutaneous flap transfer. The study of the mechanisms of injury, however, requires sophisticated experimental in vivo models. With the use of microsurgical techniques, osteomyocutaneous flap transfer can successfully be performed in rat hind limbs, allowing in vivo fluorescent microscopic analysis of post-ischemic microcirculatory dysfunction in all tissues involved, including periosteum, striated muscle, subcutis and skin. The drawback of this “acute” model is that the period of analysis is restricted to a few hours only.MethodTo overcome this limitation, the “chronic” dorsal skinfold chamber preparation, containing striated muscle and subcutis, can be used. This model allows one to study microcirculatory dysfunction after both tourniquet-induced and pressure-induced ischemia–reperfusion-induced tissue injury over a period of up to 3 weeks.ResultsWith the use of these models, recent investigations have demonstrated that ischemia–reperfusion and osteomyocutaneous flap transfer are associated with capillary perfusion failure (no-reflow), mediated by intravascular hemoconcentration, endothelial swelling and endothelin (ET)-1-mediated microvascular constriction. In addition, post-ischemic reperfusion provokes an inflammatory response (reflow paradox) in post-capillary venules, which is characterized by β2-integrin-mediated and intercellular adhesion molecule (ICAM)-1-mediated leukocyte adhesion and vascular hyperpermeability, which results in interstitial edema formation. Treatment studies have produced evidence that isovolemic hemodilution and heat shock protein induction are successful in ameliorating capillary no-reflow, while blockade of adhesion molecules, inactivation of oxygen radicals and, also, induction of heat shock proteins, are capable of reducing the post-ischemic inflammatory response.ConclusionThese experimental results not only demonstrate the importance of the use of advanced in vivo methods to delineate pathophysiological mechanisms in complex disease models, but may also provide a basis for potential prospective randomized trials to test the benefit for the patient in the daily clinical routine.

Erythropoietin reduces necrosis in critically ischemic myocutaneous tissue by protecting nutritive perfusion in a dose-dependent manner

BACKGROUND Erythropoietin (Epo), the primary regulator of erythropoiesis, has recently been shown to exert antiinflammatory and antiapoptotic properties in neuronal and myocardial tissue. We herein studied whether Epo pretreatment can reduce cell death and ischemic necrosis in a chronic in vivo model. METHODS C57BL/6 mice were treated daily for 3 consecutive days with either 500 IU EPO/kg body weight (bw) (group Epo 500, n = 8) or 5000 IU EPO/kg bw (group Epo 5000, n = 8) administered intraperitoneally 24 hours before surgery. Thereafter, a random pattern myocutaneous flap subjected to acute persistent ischemia was elevated and fixed into a dorsal skinfold chamber. Flap elevation in animals receiving the water-soluble vitamin E analog Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) served as a nonspecific antiinflammatory agent control group (Tro); untreated control animals (Con) received saline only. Capillary perfusion, leukocyte-endothelial cell interaction, apoptotic cell death, and tissue necrosis were determined over a 10-day observation period using intravital multifluorescence microscopy. RESULTS Epo 5000 (44 +/- 26 cm/cm(2)) but, more noticeably, Epo 500 (116 +/- 32 cm/cm(2)) improved capillary perfusion compared with the two control groups, particularly the Con group (9 +/- 7 cm/cm(2); P < .05). The ischemia-associated leukocytic inflammation was found drastically attenuated in both Epo-pretreatment groups. Epo 500 further decreased apoptotic cell death and was effective in significantly reducing tissue necrosis (16% +/- 4% vs Tro: 48% +/- 7% and Con: 52% +/- 4%; P < .001). No angiogenic blood vessel formation could be observed in either of the Epo groups. Of interest, Epo 5000-but not Epo 500-increased systemic hematocrit. CONCLUSION Despite the lack of neovascularization, Epo pretreatment was capable of reducing ischemic tissue necrosis by protecting capillary perfusion, ie, nutrition of the tissue. Low-dose pretreatment was more effective, a result that was most likely due to the better perfusion conditions without an increase of the hematocrit values. Thus, low-dose Epo pretreatment might represent a promising strategy to protect critically perfused ischemic tissue.

Ischemia-Induced Up-Regulation of Heme Oxygenase-1 Protects From Apoptotic Cell Death and Tissue Necrosis

BACKGROUND Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Aging is associated with an increased susceptibility to ischaemic necrosis due to microvascular perfusion failure but not a reduction in ischaemic tolerance

In the present study in a murine model of chronic ischaemia, we analysed: (i) whether aging was associated with an increased susceptibility to ischaemic necrosis, and (ii) whether this was based on microvascular dysfunction or reduced ischaemic tolerance. An ischaemic pedicled skin flap was created in the ear of homozygous hairless mice. The animals were assigned to three age groups, including adolescent (2+/-1 months), adult (10+/-2 months) and senescent (19+/-3 months). Microvascular perfusion of the ischaemic flap was assessed over 5 days by intravital microscopy, evaluating FCD (functional capillary density), capillary dilation response and the area of tissue necrosis. Expression of the stress-protein HO (haem oxygenase)-1 was determined by immunohistochemistry and Western blotting. Induction of chronic ischaemia stimulated a significant expression of HO-1 without a significant difference between the three age groups. This was associated with capillary dilation, which, however, was more pronounced in adolescent (10.5+/-2.8 microm compared with 3.95+/-0.79 microm at baseline) and adult (12.1+/-3.1 microm compared with 3.36+/-0.45 microm at baseline) animals compared with senescent animals (8.5+/-1.7 microm compared with 3.28+/-0.69 microm at baseline; P value not significant). In senescent animals, flap creation further resulted in complete cessation of capillary flow in the distal area of the flap (FCD, 0+/-0 cm/cm(2)), whereas adult (11.9+/-13.5 cm/cm(2)) and, in particular, adolescent animals (58.4+/-33.6 cm/cm(2); P<0.05) were capable of maintaining residual capillary perfusion. The age-associated microcirculatory dysfunction resulted in a significantly increased flap necrosis of 49+/-8% (P<0.05) and 42+/-8% (P<0.05) in senescent and adult animals respectively, compared with 31+/-6% in adolescent mice. Of interest, functional inhibition of HO-1 by SnPP-IX (tin protoporphyrin-IX) in adolescent mice abrogated capillary dilation, decreased functional capillary density and aggravated tissue necrosis comparably with that observed in senescent mice. Thus aging is associated with an increased susceptibility to tissue necrosis, which is due to a loss of vascular reactivity to endogenous HO-1 expression, rather than a reduction in ischaemic tolerance.

Gender-specific ischemic tissue tolerance in critically perfused skin

PurposeThe purpose of this study is to determine gender-specific differences in the development of necrosis in persistent ischemic tissue and to analyze whether differences are due to gender-specific loss of vascular reactivity or change in ischemic tolerance.MethodsHairless mice (skh-1) of both genders were assigned to three groups of adolescent, adult, and senescent age. Critical ischemia was induced by transection of the two distal pedicles of the animal’s ear. Microcirculation was assessed over a 5-day period using intravital epifluorescence microscopy. Tissue necrosis, blood flow, functional capillary density (FCD), red blood cell (RBC) velocity, and capillary diameter were analyzed.ResultsInduction of persistent ischemia caused an age-dependent demarcation of nonperfused flap tissue. Adult and senescent females developed markedly more necrosis than age-matched males (49 ± 1% vs. 37 ± 3% and 53 ± 3% vs. 44 ± 2%, respectively; p < 0.05), whereas no gender-specific difference in flap necrosis was observed in adolescent animals (31 ± 2% vs. 33 ± 3%). Gender did not affect the amount of microcirculatory dysfunction in the flap. Thus, age-matched females and males exhibited a comparable decrease of FCD, RBC velocity, and capillary dilatory response.ConclusionsBoth age and female gender may predispose for an increased susceptibility to develop ischemic tissue necrosis. The increased necrosis in female animals does not apply to an aggravated microvascular dysfunction, but rather to a reduced ischemic tissue tolerance.

Selektive Blockade des Endothelin-B-Rezeptors verbessert durch gesteigerte arterioläre Perfusion das überleben von kritisch durchbluteten muskulokutanen Lappen

Background: The insufficient perfusion of distal flap areas, which may lead to partial necrosis, still represents a major challenge in plastic and reconstructive surgery. In the process of microvascular and endothelial dysfunction, endothelins (ET) and their receptors may play an important role. The aim of the study was therefore to investigate in a chronic in vivo model the effect of various ET-receptor antagonists in critically perfused flap tissue. Methods: A random pattern musculocutaneous flap was elevated in the back of 25 C57BL/6-mice and fixed into a dorsal skinfold chamber. Repetitive intravital fluorescence microscopy was performed over a 10-day observation period to assess arteriolar diameter and blood flow (aBF), functional capillary density (FCD), the area of tissue necrosis and the development of newly formed vessels. The following substances were administered intraperitoneally 30min before induction of ischemia, as well as daily for the following 4-day period: (i) BQ-123, a specific ET-A-receptor-antagonist (ET-A, 1 mg/kg, n = 6), (ii) BQ-788, a selective ET-B-receptor-antagonist (ET-B, 1 mg/kg, n = 6) and (iii), PD-142893, a non-selective ET-AB-receptor-antagonist (ET-AB, 0,5 mg/kg, n = 6). Animals receiving saline only served as controls (n = 7). Results: Despite a slight increase of aBF within the distal flap area of controls during the 10-day observation period (3978 ± 1765pL/s; d10), a distinct restriction of FCD was noted at day 10 (32 ± 18 cm/cm2). This perfusion failure resulted in a flap necrosis of 52 ± 3 %. Selective blockade of the ET-B-receptor resulted in a dramatic increase of aBF in the distal flap area (10399 ± 5759 pl/s; d10). Thus, adequate FCD could be maintained (132 ± 42 cm/cm2; d10), resulting in a significant reduction of flap necrosis (25 ± 4 %; d10; p < 0,05 vs. control). In contrast, neither selective ET-A-receptor-blockade nor non-selective ET-AB-receptor-blockade were able to significantly affect aBF when compared to controls (ET-A: 1107 ± 1107 pl/s; ET-AB: 2436 ± 1818 pl/s; n. s.). Accordingly, flap necrosis did not differ (ET-A: 46 ± 10 %; ET-AB: 51 ± 7 %; d10) when compared with controls. Although exposed to chronic ischemia, no morphologic signs for new blood vessel formation (vascular sprouting and bud formation) could be observed in any of the experimental animals. Conclusions: The data show that only the selective ET-B-receptorantagonist is able to maintain nutritive perfusion within critically perfused flap tissue, and hence to significantly reduce flap necrosis. Accordingly, administration of ET-B-receptorantagonists may be considered in the treatment jeopardized flaps.

Endogen produziertes, jedoch nicht exogen induziertes, Stickstoffmonoxid reduziert durch Verbesserung der nutritiven Durchblutung die Nekrose in kritisch perfundierten muskulokutanen Lappen

Unsere Ergebnisse zeigen, dass endogen gebildetes Stickstoffmonoxid wesentlich an der postoperativen Mikrozirkulationsstorung in kritisch durchbluteten Arealen randomisiert perfundierter myokutaner Lappen beteiligt ist. Die zusatzliche Applikation von NO-Donoren kann lediglich die kapillare Perfusion anhaltend verbessern. Die fehlende signifikante Reduktion der Nekrose nach Arginin- Behandlung ist wohl durch den gewebeschadigenden Effekt einer vermehrten Sauerstoffradikalbildung trotz Reduktion der chronisch-ischamischen Bedingungen verursacht.